- Browse by Author
Browsing by Author "Herring, Paul"
Now showing 1 - 6 of 6
Results Per Page
Sort Options
Item Cellular & Molecular Mechanisms That Contribute to the Early Development of Skeletal Muscle & Systemic Insulin Resistance(2019-10) Grice, Brian A.; Elmendorf, Jeffrey; Considine, Robert; Herring, Paul; Mather, Kieren; Mirmira, RaghuInsulin resistance starts years before type 2 diabetes (T2D) diagnosis, even before recognition of prediabetes. Mice on a high fat diet have a similar early onset of insulin resistance, yet the mechanism remains unknown. Several studies have demonstrated that skeletal muscle insulin resistance resulting from obesity or high fat feeding does not stem from defects in proximal insulin signaling. Our lab discovered that excess plasma membrane cholesterol impairs insulin action. Excess cholesterol in the plasma membrane causes a loss of cortical actin filaments that are essential for glucose transporter GLUT4 regulation by insulin. Our cell studies further revealed that increased hexosamine biosynthesis pathway (HBP) activity increases O-linked N-acetylglucosamine modification of the transcription factor Sp1, leading to transcription of HMG-CoA reductase (HMGR), the rate-limiting enzyme in cholesterol biosynthesis. Our central hypothesis is that cholesterol accumulation mediated by HBP activity is an early reversible mechanism of high-fat diet-induced insulin resistance. We performed a series of studies and found that early high-fat feeding-induced insulin resistance is associated with a buildup of cholesterol in skeletal muscle membranes (SMM). Akin to the antidiabetic effect of caloric restriction, we found that high-fat diet removal fully mitigated SMM cholesterol accumulation and insulin resistance. Furthermore, using the cholesterol-binding agent methyl-β-cyclodextrin (MβCD), studies established causality between excess SMM cholesterol and insulin resistance. To begin to assess the role of the HBP/Sp1 in contributing to de novo cholesterol biosynthesis, SMM accumulation, and insulin resistance we treated high-fat fed mice with an Sp1 inhibitor, mithramycin. We found that mithramycin prevented SMM cholesterol accumulation and insulin resistance. This series of studies provide evidence that HBP/Sp1-mediated cholesterol accumulation in SMM is a causal, early and reversible mechanism of whole body insulin resistance.Item CRISPR-Cas9 Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin(American Heart Association, 2018-09) Lyu, Qing; Dhagia, Vidhi; Han, Yu; Guo, Bing; Wines-Samuelson, Mary E.; Christie, Christine K.; Yin, Qiangzong; Slivano, Orazio J.; Herring, Paul; Long, Xiaochun; Gupte, Sachin A.; Miano, Joseph M.; Cellular and Integrative Physiology, School of MedicineObjective- Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results- 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3×FLAG or 3×HA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a MYOCD protein product of ≈150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP (chromatin immunoprecipitation)-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. Conclusions- This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research.Item Deletion of P2Y2 receptor reveals a role for lymphotoxin-α in fatty streak formation(Elsevier, 2016-10) Aian, Shaomin; Hoggatt, April; Jones-Hall, Yava L.; Ware, Carl F.; Herring, Paul; Seye, Cheikh I.; Department of Cellular & Integrative Physiology, IU School of MedicineBackground Lymphotoxin alpha (LTα) is expressed in human atherosclerotic lesions and genetic variations in the LTα pathway have been linked to myocardial infarction. Activation of the P2Y2 nucleotide receptor (P2Y2R) regulates the production of LTα. in vitro. We aimed to uncover a potential pathway linking purinergic receptor to LTα-mediated inflammatory processes pivotal to the early stages of atherosclerosis in apolipoprotein E (ApoE−/−) deficient mice. Methods and results En face immunostaining revealed that P2Y2R and VCAM-1 are preferentially expressed in the atherosclerosis prone site of the mouse aortic sinus. Deletion of the P2Y2R gene suppresses VCAM-1 expression. Compared with ApoE−/− mice, ApoE−/− mice lacking the P2Y2R gene (ApoE−/−/P2Y2R−/−) did not develop fatty streak lesions when fed a standard chow diet for 15 weeks. Systemic and CD4+ T cell production of the pro-inflammatory cytokine lymphotoxin-alpha (LTα) were specifically inhibited in ApoE−/−/P2Y2R−/−mice. Anti-LTα preventive treatment was initiated in ApoE−/− mice with intraperitoneal administration of recombinant human tumor necrosis factor receptor 1 fusion protein (TNFR1-Fc) on 5 consecutive days before the disease onset. Remarkably, none of the TNFR1:Fc-treated ApoE−/− mice exhibited atherosclerotic lesions at any developmental stage. Significance ApoE−/− mice deficient in P2Y2R exhibit low endothelial cell VCAM-1 levels, decreased production of LTα and delayed onset of atherosclerosis. These data suggest that targeting this nucleotide receptor could be an effective therapeutic approach in atherosclerosis.Item The function of the 130kDa MLCK in regulating in vivo vascular permeability and angiogenesis(Office of the Vice Chancellor for Research, 2011-04-08) Chen, Meng; Herring, PaulDisruption of endothelial integrity is an essential component of vascular inflammation, angiogenesis, atherosclerosis, and tumor metastasis. Many studies have shown that activation of myosin light chain kinase (MLCK) in endothelial cell is correlated with increase in vascular permeability. Currently, most research in endothelial cells has focused on the 220kDa MLCK isoform which is the predominant isoform present in cultured endothelial cells. However, in freshly isolated uncultured endothelial cells, the 130kDa MLCK predominates. Yet nothing is known about the roles of the 130kDa MLCK isoform in endothelial cells. Therefore, our goal is to determine the role of the 130kDa MLCK in regulating vascular permeability and angiogenesis in vivo. To do this we will generate an endothelial cell-specific 130kDa MLCK knockout mice. As transcripts encoding the 130 and 220kDa MLCK isoforms are produced by independent promoters within the same mylk1 gene, I will selectively knockout the 130kDa MLCK by deleting unique cis-acting gene regulatory elements required for the expression of this transcript. A key element identified within the intron following the first exon of the 130kDa MLCK transcript has been flanked by LoxP sites such that Cre recombinase (Cre) mediated recombination will delete the element and attenuate expression of the 130kDa MLCK. By crossing these floxed mice with Tie2-Cre mice which express Cre specifically in endothelial cells, I will obtain endothelial cellspecific 130kDa MLCK knockout mice. In vivo vascular permeability and angiogenesis assays on these mice will allow me to determine the role played by the 130kDa MLCK in these processes. This study will not only help to identify specific functions of the 130kDa MLCK isoform, but also determine if this is a drug target for developing novel treatments of vascular diseases and cancer.Item The Regulation of MicroRNAs by Brahma-Related Gene 1 in Smooth Muscle Cells(Office of the Vice Chancellor for Research, 2013-04-05) Chen, Meng; Herring, PaulMicroRNAs (miRs) regulate the phenotypic switch of smooth muscle cells (SMCs) that occurs under several pathological conditions such as atherosclerosis. However, little is known about the transcriptional and epigenetic regulation of miR expression in SMCs. To identify miRs that are regulated by the Brahmarelated gene 1 (Brg1)-containing SWI/SNF chromatin remodeling complex we performed a microRNA array screen of RNA isolated from colonic SMCs of mice harboring a smooth muscle-specific knockout of Brg1. Quantitative RT-PCR confirmed changes in expression of several miRs, including miRs-143/145 and miR-133. Expression of dominant negative Brg1 in wild-type SMCs led to decreased expression of miRs-143/145 but not miR-133. The dominant negative Brg1 also blocked the myocardin-mediated induction of miRs-143/145 in 10T1/2 cells. Knockdown of SRF or myocardin decreased expression of miRs-143/145 in SMCs, whereas miR-133 expression was only repressed following SRF knockdown. In Brg1-null SW13 cells, miRs-143/145 but not miR-133 were dramatically induced by myocardin only in the presence of Brg1. Chromatin immunoprecipitation assays revealed that Brg1 is important for myocardin-mediated SRF binding to the miRs-143/145 promoter. Together these data show that Brg1- dependent chromatin remodeling regulates the expression of miRs-143/145 and miR-133 through distinct pathways in SMCs. This implies that chromatin remodeling complexes modulate smooth muscle phenotypes and functions not only through protein coding genes but also non-coding genes.Item The role of Brahma-related gene 1 in regulating the expression of microRNAs in colonic smooth muscle cells(Office of the Vice Chancellor for Research, 2012-04-13) Chen, Meng; Herring, PaulPrevious work by our group has shown that mice harboring null mutation of Brahma-related gene 1 (Brg1)-an ATPase subunit of SWI/SNF (SWItch/Sucrose NonFermentable) chromatin remodeling complex have reduced smooth muscle contractility and disorganized smooth muscle cells (SMCs) in colon, which are similar defects to those of microRNA maturation enzyme Dicer-deficient mice. Recently microRNAs (miRs) such as miR-143/145 have been implicated in the regulation of gene expression essential for smooth muscle cell proliferation and differentiation. Thus we aimed to identify the microRNAs that were involved in regulating the phenotypic changes in Brg1-deficient colonic smooth muscle cells and determine how Brg1 regulated them. The microRNA array screens of colonic smooth muscle and quantitative reverse transcription-polymerase chain reaction assays identified 6 miRs were down-regulated and 6 were up-regulated in smooth muscle specific Brg1 knockout tissue compared with control. Inactivation of endogenous Brg1 by introducing dominant negative Brg1 into wild type SMCs in vitro decreased miR-143/145 expression in smooth muscle cells. In Brg1 null SW13 cells, miR-143/145 were dramatically induced by myogenic transcriptional co-factor myocardin only in the presence of Brg1. Chromatin immunoprecipitation assays demonstrated that myocardin together with Brg1 increased the binding of transcription factor serum response factor (SRF) to the promoter region of miR-143/145 gene cluster. In conclusion, Brg1 together with myocardin can induce the transcription of miR-143/145 through enhancing the binding of SRF to the promoter region in SMCs. Together this suggests that SWI/SNF mediated chromatin remodeling regulates the phenotype of colonic smooth muscle by regulating expression of microRNAs that further modulate expression of their targets.