The Bromodomains of the mammalian SWI/SNF (mSWI/SNF) ATPases Brahma (BRM) and Brahma Related Gene 1 (BRG1) promote chromatin interaction and are critical for skeletal muscle differentiation [preprint]

Skeletal muscle differentiation induces changes in the epigenome of myoblasts as they proceed towards a myogenic phenotype. mSWI/SNF chromatin remodeling enzymes coordinate with lineage-determining transcription factors and are key regulators of differentiation. Three mSWI/SNF proteins, the mutually exclusive ATPases, BRG1 and BRM, and the BAF180 protein (Polybromo1, PBRM1) contain bromodomains belonging to the same structural subfamily. Bromodomains bind to acetylated lysines on histone N-terminal tails and on other proteins. Pharmacological inhibition of mSWI/SNF bromodomain function using the selective inhibitor PFI-3 reduced differentiation, decreased expression of myogenic genes, and increased the expression of cell cycle-related genes and the number of cells that remained in the cell cycle. Knockdown of BAF180 had no effect on differentiation, suggesting that only the BRG1 and BRM bromodomains contributed to differentiation. Comparison with existing gene expression data from myoblasts subjected to knockdown of BRG1 or BRM showed that bromodomain function was required for a subset of BRG1- and BRM-dependent gene expression. ChIP analysis revealed decreased BRG1 and BRM binding to target gene promoters, indicating that the BRG1 and BRM bromodomains promote chromatin binding. Thus mSWI/SNF ATPase bromodomains contribute to cell cycle exit, to skeletal muscle-specific gene expression, and to stable promoter binding by the mSWI/SNF ATPases

Selective Targeting of Bromodomains of the Bromodomain-PHD Fingers Family Impairs Osteoclast Differentiation

Histone acetyltransferases of the MYST family are recruited to chromatin by BRPF scaffolding proteins. We explored functional consequences and the therapeutic potential of inhibitors targeting acetyl-lysine dependent protein interaction domains (bromodomains) present in BRPF1-3 in bone maintenance. We report three potent and selective inhibitors: one (PFI-4) with high selectivity for the BRPF1B isoform and two pan-BRPF bromodomain inhibitors (OF-1, NI-57). The developed inhibitors displaced BRPF bromodomains from chromatin and did not inhibit cell growth and proliferation. Intriguingly, the inhibitors impaired RANKL-induced differentiation of primary murine bone marrow cells and human primary monocytes into bone resorbing osteoclasts by specifically repressing transcriptional programs required for osteoclastogenesis. The data suggest a key role of BRPF in regulating gene expression during osteoclastogenesis, and the excellent druggability of these bromodomains may lead to new treatment strategies for patients suffering from bone loss or osteolytic malignant bone lesions

The Bromodomains of the mammalian SWI/SNF (mSWI/SNF) ATPases Brahma (BRM) and Brahma Related Gene 1 (BRG1) promote chromatin interaction and are critical for skeletal muscle differentiation

Skeletal muscle regeneration is mediated by myoblasts that undergo epigenomic changes to establish the gene expression program of differentiated myofibers. mSWI/SNF chromatin remodeling enzymes coordinate with lineage-determining transcription factors to establish the epigenome of differentiated myofibers. Bromodomains bind to acetylated lysines on histone N-terminal tails and other proteins. The mutually exclusive ATPases of mSWI/SNF complexes, BRG1 and BRM, contain bromodomains with undefined functional importance in skeletal muscle differentiation. Pharmacological inhibition of mSWI/SNF bromodomain function using the small molecule PFI-3 reduced differentiation in cell culture and in vivo through decreased myogenic gene expression, while increasing cell cycle-related gene expression and the number of cells remaining in the cell cycle. Comparative gene expression analysis with data from myoblasts depleted of BRG1 or BRM showed that bromodomain function was required for a subset of BRG1- and BRM-dependent gene expression. Reduced binding of BRG1 and BRM after PFI-3 treatment showed that the bromodomain is required for stable chromatin binding at target gene promoters to alter gene expression. Our findings demonstrate that mSWI/SNF ATPase bromodomains permit stable binding of the mSWI/SNF ATPases to promoters required for cell cycle exit and establishment of muscle-specific gene expression

Calcineurin broadly regulates the initiation of skeletal muscle-specific gene expression by binding target promoters and facilitating the interaction of the SWI/SNF chromatin remodeling enzyme

Calcineurin (Cn) is a calcium-activated serine/threonine protein phosphatase that is broadly implicated in diverse cellular processes, including the regulation of gene expression. During skeletal muscle differentiation, Cn activates the NFAT transcription factor but also promotes differentiation by counteracting the negative influences of protein kinase C beta (PKCbeta) via dephosphorylation and activation of BRG1, an enzymatic subunit of the mammalian SWI/SNF ATP-dependent chromatin remodeling enzyme. Here we identified four major temporal patterns of Cn-dependent gene expression in differentiating myoblasts and determined that Cn is broadly required for the activation of the myogenic gene expression program. Mechanistically, Cn promotes gene expression through direct binding to myogenic promoter sequences and facilitating the binding of BRG1, other SWI/SNF subunit proteins, and MyoD, a critical lineage determinant for skeletal muscle differentiation. We conclude that the Cn phosphatase directly impacts the expression of myogenic genes by promoting ATP-dependent chromatin remodeling and formation of transcription-competent promoters

TRAFD1 (FLN29) Interacts with Plekhm1 and Regulates Osteoclast Acidification and Resorption

Plekhm1 is a large, multi-modular, adapter protein implicated in osteoclast vesicle trafficking and bone resorption. In patients, inactivating mutations cause osteopetrosis, and gain-of-function mutations cause osteopenia. Investigations of potential Plekhm1 interaction partners by mass spectrometry identified TRAFD1 (FLN29), a protein previously shown to suppress toll-like receptor signaling in monocytes/macrophages, thereby dampening inflammatory responses to innate immunity. We mapped the binding domains to the TRAFD1 zinc finger (aa 37-60), and to the region of Plekhm1 between its second pleckstrin homology domain and its C1 domain (aa 784-986). RANKL slightly increased TRAFD1 levels, particularly in primary osteoclasts, and the co-localization of TRAFD1 with Plekhm1 also increased with RANKL treatment. Stable knockdown of TRAFD1 in RAW 264.7 cells inhibited resorption activity proportionally to the degree of knockdown, and inhibited acidification. The lack of acidification occurred despite the presence of osteoclast acidification factors including carbonic anhydrase II, a3-V-ATPase, and the ClC7 chloride channel. Secretion of TRAP and cathepsin K were also markedly inhibited in knockdown cells. Truncated Plekhm1 in ia/ia osteopetrotic rat cells prevented vesicle localization of Plekhm1 and TRAFD1. We conclude that TRAFD1, in association with Plekhm1/Rab7-positive late endosomes-early lysosomes, has a previously unknown role in vesicle trafficking, acidification, and resorption in osteoclasts

Characteristics of dietary supplements containing in its composition glutathione (GSH) available in Poland

Glutathione (GSH) is a endogenous , low molecular weight thiol compound. It has a wide spectrum of biological activity in the body. It is an important element of the antioxidant system that protects cells against the effects of oxidative stress. As an antioxidant, GSH inactivates free radicals and reactive forms of oxygen and nitrogen in enzymatic and non-enzymatic reactions, regenerates other antioxidants, e.g. vitamins C and E, maintains - SH groups in proteins in a reduced state, participates in the detoxification of xenobiotics. The decrease in GSH concentration occurs in many diseases and in the aging process. Increasing the level of GSH in the body's cells is possible by consuming dietary components containing GSH or amino acids (especially cysteine) for its endogenous synthesis and supplementation with GSH pharmaceutical preparations. The aim of the study was to characterize dietary supplements available in Poland containing glutathione (GSH). The analysis covered 39 supplements from GSH, which were available in Poland. Their characteristics include: place / country of production; type of preparations (single and multi-component); bioavailability of GSH contained therein - preparations in the liposome formula (lipophilic GSH) and in the non-liposome formula (non-lipophilic GSH); pharmaceutical form in which these preparations were available on the commercial offer and GSH content in supplements. Among 39 GSH supplements, there were 24 single-component and 15 multi-component preparations. The largest number of GSH supplements available in Poland came from the USA and Great Britain. Among GSH supplements, there were 15 liposome (lipophilic) formula preparations, of which 12 were lipophilic one-component and 3 multi-component and 24 non-liposome (non-lipophilic) formula preparations - 12 single and 12 multi-component. Capsules, gel, liquid and tablets were the most common pharmaceutical form among all analyzed GSH supplements. The rarest pharmaceutical form was lozenges, aerosol, powder and ampoule. Analysis of GSH supplements available in Poland showed that in one single dose of the preparation was from 18 mg to 750 mg GSH. The most common dose was 250, 450 and 500 mg GSH in a single dose. One-component supplements most often contained 450 mg, then 250 and 500 mg GSH, while multi-component supplements - 250 mg GSH.Glutation (GSH) jest niskocząsteczkowym związkiem tiolowym, który wykazuje szerokie spektrum biologicznej aktywności. Jest ważnym elementem systemu antyoksydacyjnego chroniącego komórki przed skutkami stresu oksydacyjnego. Jako antyoksydant GSH unieczynnia wolne rodniki i reaktywne formy tlenu i azotu, regeneruje inne antyoksydanty np. witaminę C i E, utrzymuje grupy –SH w białkach w stanie zredukowanym, bierze udział w detoksykacji ksenobiotyków. Obniżenie stężenia GSH występuje w wielu schorzeniach. Zwiększenie poziomu GSH w komórkach organizmu jest możliwe poprzez spożywanie składników diety zawierających GSH, czy aminokwasy (szczególnie cysteina) do jego endogennej syntezy oraz suplementację preparatami farmaceutycznymi z GSH. Celem pracy była charakterystyka dostępnych w Polsce suplementów diety zawierających w swoim składzie glutation (GSH). Analizą objęto 39 suplementów z GSH. Ich charakterystyka uwzględnia: miejsce/kraj ich produkcji; typ preparatów (jedno- i wieloskładnikowe); biodostępność zawartego w nich GSH - preparaty w formule liposomowej (lipofilny GSH) i w formule nieliposomowej (nielipofilny GSH); postać farmaceutyczną preparatów oraz zawartość GSH w suplementach. Wśród 39 suplementów z GSH było 24 preparaty jedno- i 15 wieloskładnikowych. Największa liczba suplementów z GSH pochodziła z USA i Wielkiej Brytanii. Wśród suplementów z GSH było: 15 preparatów formuły liposomowej (lipofilnej ), z czego lipofilnych jednoskładnikowych było 12, a wieloskładnikowych 3 oraz 24 preparaty formuły nieliposomowej (nielipofilnej) - 12 jedno- i 12 wieloskładnikowych. Najczęściej występującą postacią farmaceutyczną wśród wszystkich analizowanych suplementów z GSH były kapsułki, żel, płyn i tabletki. W pojedynczej dawce preparatu znajdowało się od 18 mg do 750 mg GSH. Najczęściej znajdowało się 250, 450 i 500 mg GSH . Suplementy jednoskładnikowe najczęściej zawierały 450 mg GSH, natomiast wieloskładnikowe 250 mg GSH

Hydrolysis of cyclic GMP in rat peritoneal macrophages.

Intact rat peritoneal macrophages (rPM) treated with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases (PDEs), accumulated more cGMP than untreated cells. A PDE activity toward [3H]cGMP was detected in the soluble and particulate fractions of rPM. The hydrolysis of cGMP was Ca2+/calmodulin-independent but increased in the presence of cGMP excess. Similar results were obtained when [3H]cAMP was used as a substrate. The hydrolytic activity towards both nucleotides was inhibited in the presence of IBMX. Therefore, the PDEs of families 2, 5, 10 and 11 are potential candidates for cGMP hydrolysis in the rPM. They may not only regulate the cGMP level in a feedback-controlled way but also link cGMP-dependent pathways with those regulated by cAMP

Metabolism of cyclic GMP in peritoneal macrophages of rat and guinea pig.

The aim of our studies was to establish which enzymes constitute the "cGMP pathway" in rat and guinea pig peritoneal macrophages (PM). We found that in guinea pig PM synthesis of the nucleotide was significantly enhanced in response to activators of soluble guanylyl cyclase (sGC) and it was only slightly stimulated by specific activators of particulate guanylyl cyclases (pGC). In contrast, rat PM responded strongly to atrial natriuretic peptide (ANP), the activator of pGC type A. The rat cells synthesized about three-fold more cGMP than an equal number of the guinea pig cells. The activity of phosphodiesterases (PDE) hydrolyzing cGMP was apparently regulated by cGMP itself in PM of both species and again it was higher in the rat cells than in those isolated from guinea pig. However, guinea pig PM revealed an activity of Ca2+/calmodulin-dependent PDE1, which was absent in the rat cells. Using Western blotting analysis we were unable to detect the presence of cGMP-dependent protein kinase 1 (PKG1) in PM isolated from either species. In summary, our findings indicate that particulate GC-A is the main active form of GC in the rat PM, while in guinea pig macrophages the sGC activity dominates. Since the profiles of the PDE activities in rat and guinea pig PM are also different, we conclude that the mechanisms regulating cGMP metabolism in PM are species-specific. Moreover, our results suggest that targets for cGMP other than PKG1 should be present in PM of both species