AIMS: The objective of this study was to investigate the association and combined prognostic significance of the PD-L1, Smoothened protein and β-catenin expressions in patients with clear cell renal cell carcinoma (ccRCC). METHODS: The PD-L1, Smoothened protein and β-catenin expression were evaluated in 104 ccRCC patients. All studied tumor samples were acquired from nephrectomy specimens of primary tumors and not from biopsies or metastases. An indirect immunohistochemistry using polyclonal rabbit anti-Smoothened antibody, monoclonal mouse anti-human β-catenin-1 antibody, immunohistochemical assay PD-L1 28-8 pharmDx using monoclonal rabbit anti-PD-L1 antibody and anti-VHL (C- terminal) rabbit antibody was used. Immunohistochemistry was scored semiquantitavely. RESULTS: Median overall survival (OS) was significantly better in patients with lower PD-L1 expression (≤5%), Smoothened protein (SMO) expression (<5%) or cytoplasmic β-catenin expression (≤75%) than in patients with higher expressions of these biomarkers (P<0.001, P=0.047, and P<0.001, respectively). Membranous β-catenin showed an opposite effect with its lower expression (≤75%) being associated with longer OS (P=0.020). There was significant association between PD-1 and PD-L1 expression (P=0.007) and significant association of tumor grade (WHO 2016) with membranous β-catenin (P<0.001), cytoplasmic β-catenin (P=0.005), pVHL (P=0.042), PD-L1 (P=0.049) and PD-1 (P=0.028) expression. CONCLUSION: The present study provides the first data on the potential association and combined prognostic significance of frequency of primary cilia, PD-L1, Smoothened protein and β-catenin expression with the outcome in clear cell renal cell carcinoma.

• Klíčová slova
• clear cell renal carcinoma, cytoplasmic β-catenin, membranous β-catenin, primary cilia, programmed cell death protein ligand 1, smoothened protein,
• MeSH
• antigeny CD274 * metabolismus   MeSH
• beta-katenin * metabolismus   MeSH
• cilie * metabolismus   patologie   MeSH
• dospělí MeSH
• imunohistochemie MeSH
• karcinom z renálních buněk * metabolismus   mortalita   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové biomarkery metabolismus   MeSH
• nádory ledvin * metabolismus   mortalita   patologie   MeSH
• prognóza MeSH
• receptor Smoothened metabolismus   MeSH
• receptory spřažené s G-proteiny * metabolismus   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD274 * MeSH
• beta-katenin * MeSH
• CD274 protein, human MeSH Prohlížeč
• CTNNB1 protein, human MeSH Prohlížeč
• nádorové biomarkery MeSH
• receptor Smoothened MeSH
• receptory spřažené s G-proteiny * MeSH
• SMO protein, human MeSH Prohlížeč

Neural stem cells are fundamental to development of the central nervous system (CNS)-as well as its plasticity and regeneration-and represent a potential tool for neuro transplantation therapy and research. This study is focused on examination of the proliferation dynamic and fate of embryonic neural stem cells (eNSCs) under differentiating conditions. In this work, we analyzed eNSCs differentiating alone and in the presence of sonic hedgehog (SHH) or triiodothyronine (T3) which play an important role in the development of the CNS. We found that inhibition of the SHH pathway and activation of the T3 pathway increased cellular health and survival of differentiating eNSCs. In addition, T3 was able to increase the expression of the gene for the receptor smoothened (Smo), which is part of the SHH signaling cascade, while SHH increased the expression of the T3 receptor beta gene (Thrb). This might be the reason why the combination of SHH and T3 increased the expression of the thyroxine 5-deiodinase type III gene (Dio3), which inhibits T3 activity, which in turn affects cellular health and proliferation activity of eNSCs.

• Klíčová slova
• cell differentiation, embryonic neural stem cells, sonic hedgehog, triiodothyronine,
• MeSH
• jodidperoxidasa genetika   metabolismus   MeSH
• kultivované buňky MeSH
• myší embryonální kmenové buňky cytologie   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nervové kmenové buňky cytologie   metabolismus   MeSH
• neurogeneze * MeSH
• proteiny hedgehog genetika   metabolismus   MeSH
• receptor Smoothened genetika   metabolismus   MeSH
• trijodthyronin metabolismus   MeSH
• tyreoidální hormony, receptory beta genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• iodothyronine deiodinase type III MeSH Prohlížeč
• jodidperoxidasa MeSH
• proteiny hedgehog MeSH
• receptor Smoothened MeSH
• Shh protein, mouse MeSH Prohlížeč
• Smo protein, mouse MeSH Prohlížeč
• trijodthyronin MeSH
• tyreoidální hormony, receptory beta MeSH

The Hedgehog (Hh) signaling pathway is crucial for vertebrate embryonic development, tissue homeostasis and regeneration. Hh signaling is upregulated in basal cell carcinoma and medulloblastoma and Hh pathway inhibitors targeting the Smoothened (SMO) protein are in clinical use. However, the signaling cascade is incompletely understood and novel druggable proteins in the pathway are in high demand. We describe the discovery of the Hh-pathway modulator Pipinib by means of cell-based screening. Target identification and validation revealed that Pipinib selectively inhibits phosphatidylinositol 4-kinase IIIβ (PI4KB) and suppresses GLI-mediated transcription and Hh target gene expression by impairing SMO translocation to the cilium. Therefore, inhibition of PI4KB and, consequently, reduction in phosphatidyl-4-phosphate levels may be considered an alternative approach to inhibit SMO function and thus, Hedgehog signaling.

• Klíčová slova
• Hedgehog signaling, PI4KB, biological activity, inhibitors,
• MeSH
• buněčné linie MeSH
• cilie metabolismus   MeSH
• exprese genu účinky léků   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem antagonisté a inhibitory   genetika   metabolismus   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• morfoliny farmakologie   MeSH
• myši MeSH
• osteogeneze účinky léků   MeSH
• proteiny hedgehog antagonisté a inhibitory   genetika   metabolismus   MeSH
• protinádorové látky chemie   farmakologie   MeSH
• puriny farmakologie   MeSH
• receptor Smoothened genetika   metabolismus   MeSH
• RNA interference MeSH
• signální transdukce účinky léků   MeSH
• thiofeny chemie   farmakologie   MeSH
• vedlejší histokompatibilní antigeny genetika   metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• malá interferující RNA MeSH
• morfoliny MeSH
• phosphatidylinositol phosphate 4-kinase MeSH Prohlížeč
• proteiny hedgehog MeSH
• protinádorové látky MeSH
• puriny MeSH
• purmorphamine MeSH Prohlížeč
• receptor Smoothened MeSH
• thiofeny MeSH
• vedlejší histokompatibilní antigeny MeSH

OBJECTIVES: Hedgehog signalling plays a critical role during the pathogenesis of fibrosis in systemic sclerosis (SSc). Besides canonical hedgehog signalling with smoothened (SMO)-dependent activation of GLI transcription factors, GLI can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways). Here, we aimed to evaluate the role of non-canonical hedgehog signalling in SSc and to test the efficacy of direct GLI inhibitors that target simultaneously canonical and non-canonical hedgehog pathways. METHODS: The GLI inhibitor GANT-61 was used to inhibit canonical as well as non-canonical hedgehog signalling, while the SMO inhibitor vismodegib was used to selectively target canonical hedgehog signalling. Furthermore, GLI2 was selectively depleted in fibroblasts using the Cre-LoxP system. The effects of pharmacological or genetic of GLI2 on transforming growth factor-β (TGF-β) signalling were analysed in cultured fibroblasts, in bleomycin-induced pulmonary fibrosis and in mice with overexpression of a constitutively active TGF-β receptor I. RESULTS: TGF-β upregulated GLI2 in a Smad3-dependent manner and induced nuclear accumulation and DNA binding of GLI2. Fibroblast-specific knockout of GLI2 protected mice from TBRact-induced fibrosis. Combined targeting of canonical and non-canonical hedgehog signalling with direct GLI inhibitors exerted more potent antifibrotic effects than selective targeting of canonical hedgehog signalling with SMO inhibitors in experimental dermal and pulmonary fibrosis. CONCLUSIONS: Our data demonstrate that hedgehog pathways and TGF-β signalling both converge to GLI2 and that GLI2 integrates those signalling to promote tissue fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development.

• Klíčová slova
• Fibroblasts, Systemic Sclerosis, Treatment,
• MeSH
• anilidy farmakologie   MeSH
• dospělí MeSH
• fibroblasty účinky léků   metabolismus   MeSH
• fibróza MeSH
• genový knockout MeSH
• inhibitor aktivátoru plazminogenu 1 genetika   MeSH
• kolagen typu I genetika   MeSH
• kultivované buňky MeSH
• kůže účinky léků   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• mladý dospělý MeSH
• myši knockoutované MeSH
• myši transgenní MeSH
• myši MeSH
• plicní fibróza chemicky indukované   metabolismus   MeSH
• protein Gli2 s motivem zinkových prstů MeSH
• protein Smad3 metabolismus   MeSH
• protein-serin-threoninkinasy antagonisté a inhibitory   genetika   MeSH
• proteiny hedgehog metabolismus   MeSH
• pteridiny farmakologie   MeSH
• pyridiny farmakologie   MeSH
• pyrimidiny farmakologie   MeSH
• receptor Smoothened antagonisté a inhibitory   MeSH
• receptory transformujícího růstového faktoru beta antagonisté a inhibitory   genetika   MeSH
• rekombinantní proteiny farmakologie   MeSH
• růstový faktor pojivové tkáně genetika   MeSH
• senioři MeSH
• signální transdukce účinky léků   MeSH
• systémová sklerodermie genetika   metabolismus   MeSH
• TGF-beta receptor I. typu MeSH
• transformující růstový faktor beta metabolismus   farmakologie   MeSH
• transkripční faktory Krüppel-like antagonisté a inhibitory   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• myši MeSH
• senioři MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• anilidy MeSH
• GANT 61 MeSH Prohlížeč
• Gli2 protein, mouse MeSH Prohlížeč
• HhAntag691 MeSH Prohlížeč
• inhibitor aktivátoru plazminogenu 1 MeSH
• kolagen typu I MeSH
• messenger RNA MeSH
• protein Gli2 s motivem zinkových prstů MeSH
• protein Smad3 MeSH
• protein-serin-threoninkinasy MeSH
• proteiny hedgehog MeSH
• pteridiny MeSH
• pyridiny MeSH
• pyrimidiny MeSH
• receptor Smoothened MeSH
• receptory transformujícího růstového faktoru beta MeSH
• rekombinantní proteiny MeSH
• růstový faktor pojivové tkáně MeSH
• SD-208 MeSH Prohlížeč
• TGF-beta receptor I. typu MeSH
• transformující růstový faktor beta MeSH
• transkripční faktory Krüppel-like MeSH