AMP-Activated Protein Kinase-Regulated Activation of the PGC-1α Promoter in Skeletal Muscle Cells

The mechanisms by which PGC-1α gene expression is controlled in skeletal muscle remains largely undefined. Thus, we sought to investigate the transcriptional regulation of PGC-1α using AICAR, an activator of AMPK, that is known to increase PGC-1α expression. A 2.2 kb fragment of the human PGC-1α promoter was cloned and sequence analysis revealed that this TATA-less sequence houses putative consensus sites including a GC-box, a CRE, several IRSs, a SRE, binding sites for GATA, MEF2, p 53, NF-κB, and EBox binding proteins. AMPK activation for 24 hours increased PGC-1α promoter activity with concomitant increases in mRNA expression. The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at −495 within the PGC-1α promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding. Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect. Supershift analyses identified USF-1 as a DNA binding transcription factor potentially involved in regulating PGC-1α promoter activity, which was confirmed in vivo by ChIP. Overexpression of either GATA-4 or USF-1 alone increased the p851 PGC-1α promoter activity by 1.7- and 2.0-fold respectively, while co-expression of GATA-4 and USF-1 led to an additive increase in PGC-1α promoter activity. The USF-1-mediated increase in PGC-1α promoter activation led to similar increases at the mRNA level. Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1α gene expression. This could represent a potential therapeutic target to control PGC-1α expression in skeletal muscle

Critical role of TLR2 and MyD88 for functional response of macrophages to a group IIA-Secreted phospholipase A2 from snake venom

artículo (arbitrado) -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado. 2014The snake venom MT-III is a group IIA secreted phospholipase A2 (sPLA2) enzyme with functional and structural similarities with mammalian pro-inflammatory sPLA2s of the same group. Previously, we demonstrated that MT-III directly activates the innate inflammatory response of macrophages, including release of inflammatory mediators and formation of lipid droplets (LDs). However, the mechanisms coordinating these processes remain unclear. In the present study, by using TLR22/2 or MyD882/2 or C57BL/6 (WT) male mice, we report that TLR2 and MyD88 signaling have a critical role in MT-III-induced inflammatory response in macrophages. MT-III caused a marked release of PGE2, PGD2, PGJ2, IL-1b and IL-10 and increased the number of LDs in WT macrophages. In MT-III-stimulated TLR22/2 macrophages, formation of LDs and release of eicosanoids and cytokines were abrogated. In MyD882/2 macrophages, MT-III-induced release of PGE2, IL-1b and IL-10 was abrogated, but release of PGD2 and PGJ2 was maintained. In addition, COX-2 protein expression seen in MT-III-stimulated WT macrophages was abolished in both TLR22/2 and MyD882/2 cells, while perilipin 2 expression was abolished only in MyD882/2 cells. We further demonstrated a reduction of saturated, monounsaturated and polyunsaturated fatty acids and a release of the TLR2 agonists palmitic and oleic acid from MT-III-stimulated WT macrophages compared with WT control cells, thus suggesting these fatty acids as major messengers for MT-III-induced engagement of TLR2/MyD88 signaling. Collectively, our findings identify for the first time a TLR2 and MyD88-dependent mechanism that underlies group IIA sPLA2- induced inflammatory response in macrophages.This investigation was supported by research grants from FAPESP, Sao Paulo, Brazil (www.fapesp.br), grants 11/21341-5 and 10/06345-1, INCTTOX, Sao Paulo, Brazil (www.incttox.com.br), grant 573790/2008-6, CNPq PQ, Brazil (www.cnpq.br), grant 306920/2011-5, Brazil, Spanish Ministery of Science and Innovation, Spain (http://web.micinn.es/), grant BFU2010-18826.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Dissection of the MYCN locus in Feingold syndrome and isolated oesophageal atresia

Feingold syndrome (FS) is a syndromic microcephaly entity for which MYCN is the major disease-causing gene. We studied the expression pattern of MYCN at different stages of human embryonic development and collected a series of 17 FS and 12 isolated oesophageal atresia (IOA) cases. An MYCN gene deletion/mutation was identified in 47% of FS cases exclusively. We hypothesized that mutations or deletions of highly conserved non-coding elements (HCNEs) at the MYCN locus could lead to its misregulation and thereby to FS and/or IOA. We subsequently sequenced five HCNEs at the MYCN locus and designed a high-density tiling path comparative genomic hybridization array of 3.3 Mb at the MYCN locus. We found no mutations or deletions in this region, supporting the hypothesis of genetic heterogeneity in FS