Activin receptor-like kinases 1-7 (ALK1-7) regulate a complex network of SMAD-independent as well as SMAD-dependent signaling pathways. One of the widely used inhibitors for functional investigations of these processes, in particular for bone morphogenetic protein (BMP) signaling, is LDN-193189. However, LDN-193189 has insufficient kinome-wide selectivity complicating its use in cellular target validation assays. Herein, we report the identification and comprehensive characterization of two chemically distinct highly selective inhibitors of ALK1 and ALK2, M4K2234 and MU1700, along with their negative controls. We show that both MU1700 and M4K2234 efficiently block the BMP pathway via selective in cellulo inhibition of ALK1/2 kinases and exhibit favorable in vivo profiles in mice. MU1700 is highly brain penetrant and shows remarkably high accumulation in the brain. These high-quality orthogonal chemical probes offer the selectivity required to become widely used tools for in vitro and in vivo investigation of BMP signaling.

• MeSH
• aktivinové receptory typu I antagonisté a inhibitory   metabolismus   MeSH
• aktivinové receptory typu II * metabolismus   antagonisté a inhibitory   MeSH
• inhibitory proteinkinas farmakologie   chemie   MeSH
• kostní morfogenetické proteiny metabolismus   MeSH
• lidé MeSH
• molekulární sondy chemie   MeSH
• myši MeSH
• objevování léků MeSH
• pyrazoly chemie   farmakologie   chemická syntéza   MeSH
• signální transdukce účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Bone morphogenetic proteins (BMPs) and receptors (BMPR-1A, BMPR-1B, BMPR-2) have been shown to be vital for female reproduction, while their roles in males are poorly described. Our study was undertaken to specify the function of BMPR-1B in steroidogenic enzyme gene expression, testosterone production and reproductive development in male mice, given that Bmpr1b mRNA is expressed in mouse testis and Bmpr1b knockout results in compromised fertility. Male mice were passively immunized for 6 days with anti-BMPR-1B in the presence or absence of exogenous gonadotrophins. We then measured the effects of anti-BMPR-1B on testicular hydroxysteroid dehydrogenase isoforms (Hsd3b1, Hsd3b6, and Hsd17b3) and aromatase (Cyp19) mRNA expression, testicular and serum testosterone levels, and testis and seminal vesicle weight. In vitro testosterone production in response to anti-BMPR-1B was determined using testicular culture, and Leydig cell culture in the presence or absence of gonadotrophins. In Leydig cell culture the contribution of seminiferous tubules and Leydig cells were examined by preconditioning the media with these testicular constituents. In adult mice, anti-BMPR-1B increased testosterone and Hsd3b1 but decreased Hsd3b6 and Cyp19 mRNA. In adult testicular culture and seminiferous tubule conditioned Leydig cell culture, anti-BMPR-1B reduced testosterone, while in normal and Leydig cell conditioned Leydig cell culture it increased testosterone levels. In pubertal mice, anti-BMPR-1B reduced gonadotrophin stimulated seminal vesicle growth. In conclusion, BMPR-1B has specific developmental functions in the autocrine and paracrine regulation of testicular steroidogenic enzyme gene expression and testosterone production in adults and in the development of seminal vesicles during puberty.

• MeSH
• aromatasa metabolismus   MeSH
• exprese genu MeSH
• kostní morfogenetické proteiny metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• myši MeSH
• pohlavní dospělost MeSH
• receptory kostního morfogenetického proteinu metabolismus   MeSH
• testis * metabolismus   MeSH
• testosteron * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

OBJECTIVE: GDF11 is a member of the TGF-β superfamily that was recently implicated as potential "rejuvenating" factor, which can ameliorate metabolic disorders. The main objective of the presented study was to closely characterize the role of GDF11 signaling in the glucose homeostasis and in the differentiation of white adipose tissue. METHODS: We performed microscopy imaging, biochemical and transcriptomic analyses of adipose tissues of 9 weeks old ob/ob mice and murine and human pre-adipocyte cell lines. RESULTS: Our in vivo experiments employing GDF11 treatment in ob/ob mice showed improved glucose/insulin homeostasis, decreased weight gain and white adipocyte size. Furthermore, GDF11 treatment inhibited adipogenesis in pre-adipocytes by ALK5-SMAD2/3 activation in cooperation with the WNT/β-catenin pathway, whose inhibition resulted in adipogenic differentiation. Lastly, we observed significantly elevated levels of the adipokine hormone adiponectin and increased glucose uptake by mature adipocytes upon GDF11 exposure. CONCLUSION: We show evidence that link GDF11 to adipogenic differentiation, glucose, and insulin homeostasis, which are pointing towards potential beneficial effects of GDF11-based "anti-obesity" therapy.

• MeSH
• adipogeneze * MeSH
• adiponektin metabolismus   MeSH
• beta-katenin * metabolismus   MeSH
• buněčná diferenciace fyziologie   MeSH
• glukosa metabolismus   MeSH
• inzulin metabolismus   MeSH
• kostní morfogenetické proteiny metabolismus   MeSH
• lidé MeSH
• myši MeSH
• protein Smad2 MeSH
• protein Smad3 MeSH
• receptory regulované proteiny Smad MeSH
• růstové diferenciační faktory metabolismus   MeSH
• signální dráha Wnt MeSH
• TGF-beta receptor I. typu MeSH
• transformující růstový faktor beta metabolismus   MeSH
• tukové buňky metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Hepatocellular carcinoma (HCC) is one of the fastest-growing causes of cancer-related mortalities worldwide and this trend is mimicked by the surge of non-alcoholic fatty liver disease (NAFLD). Altered hepatic lipid metabolism promotes HCC development through inflammation and activation of oncogenes. GDF11 is a member of the TGF-β superfamily and recent data have implicated GDF11 as an anti-aging factor that can alleviate high-fat diet induced obesity, hyperglycemia, insulin resistance and NAFLD. However, its role in hepatic lipid metabolism is still not fully delineated. The aim of the present study was to characterize the role of GDF11 in hepatic and HCC cells lipid accumulation. To achieve this, we performed imaging, biochemical, lipidomic, and transcriptomic analyses in primary hepatocytes and in HCC cells treated with GDF11 to study the GDF11-activated signaling pathways. GDF11 treatment rapidly triggered ALK5-dependent SMAD2/3 nuclear translocation and elevated lipid droplets in HCC cells, but not in primary hepatocytes. In HCC cells, ALK5 inhibition hampered GDF11-mediated SMAD2/3 signaling and attenuated lipid accumulation. Using ultra-high-performance liquid chromatography/mass spectrometry, we detected increased accumulation of longer acyl-chain di/tri-acylglycerols and glycerophospholipids. Unbiased transcriptomic analysis identified TGF-β and PI3K-AKT signaling among the top pathways/cellular processes activated in GDF11 treated HCC cells. In summary, GDF11 supplementation promotes pro-lipogenic gene expression and lipid accumulation in HCC cells. Integration of our "omics" data pointed to a GDF11-induced upregulation of de novo lipogenesis through activation of ALK5/SMAD2/3/PI3K-AKT pathways. Thus, GDF11 could contribute to metabolic reprogramming and dysregulation of lipid metabolism in HCC cells, without effects on healthy hepatocytes.

• MeSH
• hepatocelulární karcinom patologie   MeSH
• hepatocyty metabolismus   MeSH
• kostní morfogenetické proteiny metabolismus   MeSH
• lidé MeSH
• lipogeneze MeSH
• metabolismus lipidů * MeSH
• nádorové buněčné linie MeSH
• nádory jater patologie   MeSH
• protein Smad2 metabolismus   MeSH
• růstové diferenciační faktory metabolismus   MeSH
• signální transdukce * MeSH
• TGF-beta receptor I. typu metabolismus   MeSH
• upregulace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Despite recent progress in recognizing the importance of mesenchymal cells for the homeostasis of the intestinal system, the current picture of how these cells communicate with the associated epithelial layer remains unclear. To describe the relevant cell populations in an unbiased manner, we carried out a single-cell transcriptome analysis of the adult murine colon, producing a high-quality atlas of matched colonic epithelium and mesenchyme. We identify two crypt-associated colonic fibroblast populations that are demarcated by different strengths of platelet-derived growth factor receptor A (Pdgfra) expression. Crypt-bottom fibroblasts (CBFs), close to the intestinal stem cells, express low levels of Pdgfra and secrete canonical Wnt ligands, Wnt potentiators, and bone morphogenetic protein (Bmp) inhibitors. Crypt-top fibroblasts (CTFs) exhibit high Pdgfra levels and secrete noncanonical Wnts and Bmp ligands. While the Pdgfralow cells maintain intestinal stem cell proliferation, the Pdgfrahigh cells induce differentiation of the epithelial cells. Our findings enhance our understanding of the crosstalk between various colonic epithelial cells and their associated mesenchymal signaling hubs along the crypt axis-placing differential Pdgfra expression levels in the spotlight of intestinal fibroblast identity.

• MeSH
• analýza jednotlivých buněk metody   MeSH
• buněčná diferenciace fyziologie   MeSH
• epitelové buňky metabolismus   MeSH
• fibroblasty klasifikace   metabolismus   MeSH
• homeostáza MeSH
• kmenové buňky cytologie   MeSH
• kolon metabolismus   fyziologie   MeSH
• kostní morfogenetické proteiny metabolismus   MeSH
• mezoderm cytologie   fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• proliferace buněk fyziologie   MeSH
• signální transdukce MeSH
• stanovení celkové genové exprese metody   MeSH
• střeva fyziologie   MeSH
• střevní sliznice metabolismus   MeSH
• transkriptom genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND & AIMS: Growth Differentiation Factor 11 (GDF11) is an anti-aging factor, yet its role in liver diseases is not established. We evaluated the role of GDF11 in healthy conditions and in the transition from non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH). RESULTS: GDF11 mRNA levels positively correlated with NAFLD activity score and with CPT1, SREBP, PPARγ and Col1A1 mRNA levels, and associated to portal fibrosis, in morbidly obese patients with NAFLD/NASH. GDF11-treated mice showed mildly exacerbated hepatic collagen deposition, accompanied by weight loss and without changes in liver steatosis or inflammation. GDF11 triggered ALK5-dependent SMAD2/3 nuclear translocation and the pro-fibrogenic activation of HSC. CONCLUSIONS: GDF11 supplementation promotes mild liver fibrosis. Even considering its beneficial metabolic effects, caution should be taken when considering therapeutics that regulate GDF11. METHODS: We analyzed liver biopsies from a cohort of 33 morbidly obese adults with NAFLD/NASH. We determined the correlations in mRNA expression levels between GDF11 and genes involved in NAFLD-to-NASH progression and with pathological features. We also exposed wild type or obese mice with NAFLD to recombinant GDF11 by daily intra-peritoneal injection and monitor the hepatic pathological changes. Finally, we analyzed GDF11-activated signaling pathways in hepatic stellate cells (HSC).

• MeSH
• buněčné linie MeSH
• dospělí MeSH
• experimentální cirhóza jater chemicky indukované   metabolismus   patologie   MeSH
• jaterní cirhóza diagnóza   etiologie   genetika   metabolismus   MeSH
• jaterní hvězdicovité buňky metabolismus   patologie   MeSH
• játra metabolismus   patologie   MeSH
• kostní morfogenetické proteiny genetika   metabolismus   toxicita   MeSH
• lidé středního věku MeSH
• lidé MeSH
• morbidní obezita komplikace   diagnóza   MeSH
• myši inbrední C57BL MeSH
• nealkoholová steatóza jater diagnóza   etiologie   genetika   metabolismus   MeSH
• progrese nemoci MeSH
• růstové diferenciační faktory genetika   metabolismus   toxicita   MeSH
• signální transdukce MeSH
• studie případů a kontrol MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Wnt and BMP signaling pathways are two key molecular machineries regulating development and homeostasis. The efficient coordination of Wnt and BMP is essential in many developmental processes such as establishment of antero-posterior and dorso-ventral body axis, regulation of convergent extension, or development of various organ systems. SMAD ubiquitination regulatory factor (Smurf) family of E3 ubiquitin ligases are important and evolutionary conserved regulators of TGF-β/BMP signaling pathways. Smurf2 has been previously shown to regulate Wnt/planar cell polarity (PCP) signaling pathway by ubiquitinating Prickle1, one of the key components of PCP. We explored the role of Smurf2 in Wnt pathways in further detail and identified that Smurf2 is also a ubiquitin ligase of Dishevelled (DVL), the key cytoplasmic signal transducer in the Wnt pathway. Interestingly, the Smurf2 and DVL relationship expands beyond substrate-E3 ligase. We can show that DVL activates Smurf2, which allows Smurf2 to ubiquitinate its substrates from Wnt/PCP (Prickle1) as well as TGF-β/BMP (Smad2) pathways more efficiently. Using SMAD7 as an example of Smurf2 activator we show that DVL and SMAD7 both activates Smurf2 activity. In HEK293 cells the deficiency of DVL phenocopies absence of Smurf2 and leads to the increased phosphorylation of R-Smads. Smurf2-DVL connection provides a novel and intriguing point of crosstalk for Wnt and BMP pathways.

• MeSH
• biologické modely MeSH
• HEK293 buňky MeSH
• kostní morfogenetické proteiny metabolismus   MeSH
• lidé MeSH
• nádorové supresorové proteiny metabolismus   MeSH
• protein dishevelled metabolismus   MeSH
• proteiny s doménou LIM metabolismus   MeSH
• proteolýza MeSH
• signální dráha Wnt * MeSH
• signální transdukce MeSH
• transformující růstový faktor beta metabolismus   MeSH
• ubikvitinace MeSH
• ubikvitinligasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The BMP signaling pathway has been shown to be involved in different aspects of embryonic development across diverse metazoan phyla. Comparative studies on the roles of the BMP signaling pathway provide crucial insights into the evolution of the animal body plans. In this chapter, we present the general workflow on how to investigate the roles of BMP signaling pathway during amphioxus embryonic development. As amphioxus are basal invertebrate chordates, studies on the BMP signaling pathway in amphioxus could elucidate the functional evolution of BMP pathway in the chordate group. Here, we describe methods for animal husbandry, spawning induction, and manipulation of the BMP signaling pathway during embryonic development through drug inhibitors and recombinant proteins. We also introduce an efficient method of using mesh baskets to handle amphioxus embryos for fluorescence immunostaining and multicolor fluorescence in situ hybridization and to assay the effects of manipulating BMP signaling pathway during amphioxus embryogenesis.

• MeSH
• biologické modely * MeSH
• embryo nesavčí MeSH
• embryonální vývoj MeSH
• hybridizace in situ fluorescenční MeSH
• kopinatci metabolismus   MeSH
• kostní morfogenetické proteiny metabolismus   farmakologie   MeSH
• rekombinantní proteiny farmakologie   MeSH
• signální transdukce * účinky léků   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Establishment of asymmetry along the left-right (LR) body axis in vertebrates requires interplay between Nodal and Bmp signaling pathways. In the basal chordate amphioxus, the left-sided activity of the Nodal signaling has been attributed to the asymmetric morphogenesis of paraxial structures and pharyngeal organs, however the role of Bmp signaling in LR asymmetry establishment has not been addressed to date. Here, we show that Bmp signaling is necessary for the development of LR asymmetric morphogenesis of amphioxus larvae through regulation of Nodal signaling. Loss of Bmp signaling results in loss of the left-sided expression of Nodal, Gdf1/3, Lefty and Pitx and in gain of ectopic expression of Cerberus on the left side. As a consequence, the larvae display loss of the offset arrangement of axial structures, loss of the left-sided pharyngeal organs including the mouth, and ectopic development of the right-sided organs on the left side. Bmp inhibition thus phenocopies inhibition of Nodal signaling and results in the right isomerism. We conclude that Bmp and Nodal pathways act in concert to specify the left side and that Bmp signaling plays a fundamental role during LR development in amphioxus.

• MeSH
• embryo nesavčí cytologie   embryologie   MeSH
• faktory určující pravo-levou symetrii biosyntéza   MeSH
• kopinatci cytologie   embryologie   MeSH
• kostní morfogenetické proteiny metabolismus   MeSH
• protein nodal metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• vývojová regulace genové exprese fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The full maturational capability of mammalian oocytes is accompanied by nuclear and cytoplasmic modifications, which are associated with proliferation and differentiation of surrounding cumulus cells. These events are regulated on molecular level by the expression of target genes involved in signal transduction pathways crucial for folliculogenesis and oogenesis. Transforming growth factor beta signaling includes several molecules that are involved in the regulation of oogenesis and embryo growth, including bone morphogenetic protein (BMP). However, the BMP-related gene expression profile in oocytes at different maturational stages requires further investigation. METHODS: Oocytes were isolated from pubertal crossbred Landrace gilts follicles, selected with a use of BCB staining test and analyzed before and after in vitro maturation. Gene expression profiles were examined using an Affymetrix microarray approach and validated by RT-qPCR. Database for Annotation, Visualization, and Integrated Discovery (DAVID) software was used for the extraction of the genes belonging to a BMP-signaling pathway ontology group. RESULTS: The assay revealed 12,258 different transcripts in porcine oocytes, among which 379 genes were down-regulated and 40 were up-regulated. The DAVID database indicated a "BMP signaling pathway" ontology group, which was significantly regulated in both groups of oocytes. We discovered five up-regulated genes in oocytes before versus after in vitro maturation (IVM): chordin-like 1 (CHRDL1), follistatin (FST), transforming growth factor-beta receptor-type III (TGFβR3), decapentaplegic homolog 4 (SMAD4), and inhibitor of DNA binding 1 (ID1). CONCLUSIONS: Increased expression of CHRDL1, FST, TGFβR3, SMAD4, and ID1 transcripts before IVM suggested a subordinate role of the BMP signaling pathway in porcine oocyte maturational competence. Conversely, it is postulated that these genes are involved in early stages of folliculogenesis and oogenesis regulation in pigs, since in oocytes before IVM increased expression was observed.

• MeSH
• IVM techniky * MeSH
• kostní morfogenetické proteiny genetika   metabolismus   MeSH
• kumulární buňky cytologie   metabolismus   fyziologie   MeSH
• mikročipová analýza MeSH
• oocyty cytologie   metabolismus   fyziologie   MeSH
• oogeneze genetika   MeSH
• prasata genetika   metabolismus   MeSH
• signální transdukce genetika   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH