Abnormal epigenetic changes during differentiation of human skeletal muscle stem cells from obese subjects

Contains fulltext : 169731.pdf (publisher's version ) (Open Access)BACKGROUND: Human skeletal muscle stem cells are important for muscle regeneration. However, the combined genome-wide DNA methylation and expression changes taking place during adult myogenesis have not been described in detail and novel myogenic factors may be discovered. Additionally, obesity is associated with low relative muscle mass and diminished metabolism. Epigenetic alterations taking place during myogenesis might contribute to these defects. METHODS: We used Infinium HumanMethylation450 BeadChip Kit (Illumina) and HumanHT-12 Expression BeadChip (Illumina) to analyze genome-wide DNA methylation and transcription before versus after differentiation of primary human myoblasts from 14 non-obese and 14 obese individuals. Functional follow-up experiments were performed using siRNA mediated gene silencing in primary human myoblasts and a transgenic mouse model. RESULTS: We observed genome-wide changes in DNA methylation and expression patterns during differentiation of primary human muscle stem cells (myoblasts). We identified epigenetic and transcriptional changes of myogenic transcription factors (MYOD1, MYOG, MYF5, MYF6, PAX7, MEF2A, MEF2C, and MEF2D), cell cycle regulators, metabolic enzymes and genes previously not linked to myogenesis, including IL32, metallothioneins, and pregnancy-specific beta-1-glycoproteins. Functional studies demonstrated IL-32 as a novel target that regulates human myogenesis, insulin sensitivity and ATP levels in muscle cells. Furthermore, IL32 transgenic mice had reduced insulin response and muscle weight. Remarkably, approximately 3.7 times more methylation changes (147,161 versus 39,572) were observed during differentiation of myoblasts from obese versus non-obese subjects. In accordance, DNMT1 expression increased during myogenesis only in obese subjects. Interestingly, numerous genes implicated in metabolic diseases and epigenetic regulation showed differential methylation and expression during differentiation only in obese subjects. CONCLUSIONS: Our study identifies IL-32 as a novel myogenic regulator, provides a comprehensive map of the dynamic epigenome during differentiation of human muscle stem cells and reveals abnormal epigenetic changes in obesity

Differential effects of human and plant N-acetylglucosaminyltransferase I (GnTI) in plants

In plants and animals, the first step in complex type N-glycan formation on glycoproteins is catalyzed by N-acetylglucosaminyltransferase I (GnTI). We show that the cgl1-1 mutant of Arabidopsis, which lacks GnTI activity, is fully complemented by YFP-labeled plant AtGnTI, but only partially complemented by YFP-labeled human HuGnTI and that this is due to post-transcriptional events. In contrast to AtGnTI-YFP, only low levels of HuGnTI-YFP protein was detected in transgenic plants. In protoplast co-transfection experiments all GnTI-YFP fusion proteins co-localized with a Golgi marker protein, but only limited co-localization of AtGnTI and HuGnTI in the same plant protoplast. The partial alternative targeting of HuGnTI in plant protoplasts was alleviated by exchanging the membrane-anchor domain with that of AtGnTI, but in stably transformed cgl1-1 plants this chimeric GnTI still did not lead to full complementation of the cgl1-1 phenotype. Combined, the results indicate that activity of HuGnTI in plants is limited by a combination of reduced protein stability, alternative protein targeting and possibly to some extend to lower enzymatic performance of the catalytic domain in the plant biochemical environment

STAT1 Hyperphosphorylation and Defective IL12R/IL23R Signaling Underlie Defective Immunity in Autosomal Dominant Chronic Mucocutaneous Candidiasis

We recently reported the genetic cause of autosomal dominant chronic mucocutaneous candidiasis (AD-CMC) as a mutation in the STAT1 gene. In the present study we show that STAT1 Arg274Trp mutations in the coiled-coil (CC) domain is the genetic cause of AD-CMC in three families of patients. Cloning and transfection experiments demonstrate that mutated STAT1 inhibits IL12R/IL-23R signaling, with hyperphosphorylation of STAT1 as the likely underlying molecular mechanism. Inhibition of signaling through the receptors for IL-12 and IL-23 leads to strongly diminished Th1/Th17 responses and hence to increased susceptibility to fungal infections. The challenge for the future is to translate this knowledge into novel strategies for the treatment of this severe immunodeficiency

Cytokines and microbicidal molecules regulated by IL-32 in THP-1-derived human macrophages infected with New World Leishmania species

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Interleukin 32 (IL-32) contains a typical alpha-helix bundle structure that resembles focal adhesion targeting region of focal adhesion kinase-1.

Contains fulltext : 108393.pdf (Publisher’s version ) (Open Access)IL-32 can be expressed in several isoforms. The amino acid sequences of the major IL-32 isoforms were used to predict the secondary and tertiary protein structure by I-TASSER software. The secondary protein structure revealed coils and alpha-helixes, but no beta sheets. Furthermore, IL-32 contains an RGD motif, which potentially activates procaspase-3 intracellular and or binds to integrins. Mutation of the RGD motif did not result in inhibition of the IL-32beta- or IL-32gamma-induced cytotoxicity mediated through caspase-3. Although IL-32alpha interacted with the extracellular part of alphaVbeta3 and alphaVbeta6 integrins, only the alphaVbeta3 binding was inhibited by small RGD peptides. Additionally, IL-32beta was able to bind to alphaVbeta3 integrins, whereas this binding was not inhibited by small RGD peptides. In addition to the IL-32/integrin interactions, we observed that IL-32 is also able to interact with intracellular proteins that are involved in integrin and focal adhesion signaling. Modeling of IL-32 revealed a distinct alpha-helix protein resembling the focal adhesion targeting region of focal adhesion kinase (FAK). Inhibition of FAK resulted in modulation of the IL-32beta- or IL-32gamma-induced cytotoxicity. Interestingly, IL-32alpha binds to paxillin without the RGD motif being involved. Finally, FAK inhibited IL-32alpha/paxillin binding, whereas FAK also could interact with IL-32alpha, demonstrating that IL-32 is a member of the focal adhesion protein complex. This study demonstrates for the first time that IL-32 binds to the extracellular domain of integrins and to intracellular proteins like paxillin and FAK, suggesting a dual role for IL-32 in integrin signaling

Differential function of the NACHT-LRR (NLR) members Nod1 and Nod2 in arthritis

The pathogenesis of chronic joint inflammation remains unclear, although the involvement of pathogen recognition receptors has been suggested recently. In the present article, we describe the role of two members of the NACHT-LRR (NLR) family, Nod1 (nucleotide-binding oligomerization domain) and Nod2 in a model of acute joint inflammation induced by intraarticular injection of Streptococcus pyogenes cell wall fragments. Here, we show that Nod2 deficiency resulted in reduced joint inflammation and protection against early cartilage damage. In contrast, Nod1 gene-deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage, consistent with a model in which Nod1 controls the inflammatory reaction. To explore whether the different function of Nod1 and Nod2 occurs also in humans, we exposed peripheral blood mononuclear cells (PBMCs) carrying either Nod1ins/del or Nod2fs mutation with SCW fragments in vitro. Production of both TNFα and IL-1β was clearly impaired in PBMCs carrying the Nod2fs compared with PBMCs isolated from healthy controls. In line with results in Nod1 gene-deficient mice, PBMCs from individuals bearing a newly described Nod1 mutation produced enhanced levels of proinflammatory cytokines after 24-h stimulation with SCW fragments. These data indicate that the NLR family members Nod1 and Nod2 have different functions in controlling inflammation, and that intracellular Nod1–Nod2 interactions may determine the severity of arthritis in this experimental model. Whether a distorted balance between the function of Nod1 and/or Nod2 is involved in the pathogenesis of human autoinflammatory or autoimmune disease, such as rheumatoid arthritis, remains to be elucidated