Spondylocarpotarsal syndrome (SCT) is a rare musculoskeletal disorder characterized by short stature and vertebral, carpal, and tarsal fusions resulting from biallelic nonsense mutations in the gene encoding filamin B (FLNB). Utilizing a FLNB knockout mouse, we showed that the vertebral fusions in SCT evolved from intervertebral disc (IVD) degeneration and ossification of the annulus fibrosus (AF), eventually leading to full trabecular bone formation. This resulted from alterations in the TGFβ/BMP signaling pathway that included increased canonical TGFβ and noncanonical BMP signaling. In this study, the role of FLNB in the TGFβ/BMP pathway was elucidated using in vitro, in vivo, and ex vivo treatment methodologies. The data demonstrated that FLNB interacts with inhibitory Smads 6 and 7 (i-Smads) to regulate TGFβ/BMP signaling and that loss of FLNB produces increased TGFβ receptor activity and decreased Smad 1 ubiquitination. Through the use of small molecule inhibitors in an ex vivo spine model, TGFβ/BMP signaling was modulated to design a targeted treatment for SCT and disc degeneration. Inhibition of canonical and noncanonical TGFβ/BMP pathway activity restored Flnb-/- IVD morphology. These most effective improvements resulted from specific inhibition of TGFβ and p38 signaling activation. FLNB acts as a bridge for TGFβ/BMP signaling crosstalk through i-Smads and is key for the critical balance in TGFβ/BMP signaling that maintains the IVD. These findings further our understanding of IVD biology and reveal new molecular targets for disc degeneration as well as congenital vertebral fusion disorders.

• Publikační typ
• časopisecké články MeSH

Osteogenesis imperfecta (OI) is a genetically heterogenous disorder most often due to heterozygosity for mutations in the type I procollagen genes, COL1A1 or COL1A2. The disorder is characterized by bone fragility leading to increased fracture incidence and long-bone deformities. Although multiple mechanisms underlie OI, endoplasmic reticulum (ER) stress as a cellular response to defective collagen trafficking is emerging as a contributor to OI pathogenesis. Herein, we used 4-phenylbutiric acid (4-PBA), an established chemical chaperone, to determine if treatment of Aga2+/- mice, a model for moderately severe OI due to a Col1a1 structural mutation, could attenuate the phenotype. In vitro, Aga2+/- osteoblasts show increased protein kinase RNA-like endoplasmic reticulum kinase (PERK) activation protein levels, which improved upon treatment with 4-PBA. The in vivo data demonstrate that a postweaning 5-week 4-PBA treatment increased total body length and weight, decreased fracture incidence, increased femoral bone volume fraction (BV/TV), and increased cortical thickness. These findings were associated with in vivo evidence of decreased bone-derived protein levels of the ER stress markers binding immunoglobulin protein (BiP), CCAAT/-enhancer-binding protein homologous protein (CHOP), and activating transcription factor 4 (ATF4) as well as increased levels of the autophagosome marker light chain 3A/B (LC3A/B). Genetic ablation of CHOP in Aga2+/- mice resulted in increased severity of the Aga2+/- phenotype, suggesting that the reduction in CHOP observed in vitro after treatment is a consequence rather than a cause of reduced ER stress. These findings suggest the potential use of chemical chaperones as an adjunct treatment for forms of OI associated with ER stress. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

• MeSH
• butylaminy MeSH
• fenotyp MeSH
• kolagen typu I metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• molekulární chaperony metabolismus   MeSH
• mutace MeSH
• myši MeSH
• osteoblasty metabolismus   MeSH
• osteogenesis imperfecta * farmakoterapie   genetika   metabolismus   MeSH
• osteogeneze MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Alterations in the balance between skeletogenesis and adipogenesis is a pathogenic feature in multiple skeletal disorders. Clinically, enhanced bone marrow adiposity in bones impairs mobility and increases fracture risk, reducing the quality of life of patients. The molecular mechanism that underlies the balance between skeletogenesis and adipogenesis is not completely understood but alterations in skeletal progenitor cells' differentiation pathway plays a key role. We recently demonstrated that parathyroid hormone (PTH)/PTH-related peptide (PTHrP) control the levels of DEPTOR, an inhibitor of the mechanistic target of rapamycin (mTOR), and that DEPTOR levels are altered in different skeletal diseases. Here, we show that mutations in the PTH receptor-1 (PTH1R) alter the differentiation of skeletal progenitors in two different skeletal genetic disorders and lead to accumulation of fat or cartilage in bones. Mechanistically, DEPTOR controls the subcellular localization of TAZ (transcriptional co-activator with a PDZ-binding domain), a transcriptional regulator that governs skeletal stem cells differentiation into either bone and fat. We show that DEPTOR regulation of TAZ localization is achieved through the control of Dishevelled2 (DVL2) phosphorylation. Depending on nutrient availability, DEPTOR directly interacts with PTH1R to regulate PTH/PTHrP signaling or it forms a complex with TAZ, to prevent its translocation to the nucleus and therefore inhibit its transcriptional activity. Our data point DEPTOR as a key molecule in skeletal progenitor differentiation; its dysregulation under pathologic conditions results in aberrant bone/fat balance.

• Publikační typ
• časopisecké články MeSH

Achondroplasia is the most prevalent genetic form of dwarfism in humans and is caused by activating mutations in FGFR3 tyrosine kinase. The clinical need for a safe and effective inhibitor of FGFR3 is unmet, leaving achondroplasia currently incurable. Here, we evaluated RBM-007, an RNA aptamer previously developed to neutralize the FGFR3 ligand FGF2, for its activity against FGFR3. In cultured rat chondrocytes or mouse embryonal tibia organ culture, RBM-007 rescued the proliferation arrest, degradation of cartilaginous extracellular matrix, premature senescence, and impaired hypertrophic differentiation induced by FGFR3 signaling. In cartilage xenografts derived from induced pluripotent stem cells from individuals with achondroplasia, RBM-007 rescued impaired chondrocyte differentiation and maturation. When delivered by subcutaneous injection, RBM-007 restored defective skeletal growth in a mouse model of achondroplasia. We thus demonstrate a ligand-trap concept of targeting the cartilage FGFR3 and delineate a potential therapeutic approach for achondroplasia and other FGFR3-related skeletal dysplasias.

• MeSH
• achondroplazie * farmakoterapie   genetika   MeSH
• aptamery nukleotidové * MeSH
• buněčná diferenciace MeSH
• chondrocyty MeSH
• krysa rodu rattus MeSH
• myši MeSH
• receptor fibroblastových růstových faktorů, typ 3 genetika   MeSH
• vývoj kostí MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Beyond its structural role in the skeleton, the extracellular matrix (ECM), particularly basement membrane proteins, facilitates communication with intracellular signaling pathways and cell to cell interactions to control differentiation, proliferation, migration and survival. Alterations in extracellular proteins cause a number of skeletal disorders, yet the consequences of an abnormal ECM on cellular communication remains less well understood METHODS: Clinical and radiographic examinations defined the phenotype in this unappreciated bent bone skeletal disorder. Exome analysis identified the genetic alteration, confirmed by Sanger sequencing. Quantitative PCR, western blot analyses, immunohistochemistry, luciferase assay for WNT signaling were employed to determine RNA, proteins levels and localization, and dissect out the underlying cell signaling abnormalities.  Migration and wound healing assays examined cell migration properties. FINDINGS: This bent bone dysplasia resulted from biallelic mutations in LAMA5, the gene encoding the alpha-5 laminin basement membrane protein. This finding uncovered a mechanism of disease driven by ECM-cell interactions between alpha-5-containing laminins, and integrin-mediated focal adhesion signaling, particularly in cartilage. Loss of LAMA5 altered β1 integrin signaling through the non-canonical kinase PYK2 and the skeletal enriched SRC kinase, FYN. Loss of LAMA5 negatively impacted the actin cytoskeleton, vinculin localization, and WNT signaling. INTERPRETATION: This newly described mechanism revealed a LAMA5-β1 Integrin-PYK2-FYN focal adhesion complex that regulates skeletogenesis, impacted WNT signaling and, when dysregulated, produced a distinct skeletal disorder. FUNDING: Supported by NIH awards R01 AR066124, R01 DE019567, R01 HD070394, and U54HG006493, and Czech Republic grants INTER-ACTION LTAUSA19030, V18-08-00567 and GA19-20123S.

• MeSH
• alely * MeSH
• buněčná adheze genetika   MeSH
• chondrocyty metabolismus   MeSH
• fenotyp MeSH
• fokální adhezní kinasa 2 genetika   metabolismus   MeSH
• genetická predispozice k nemoci MeSH
• genetické asociační studie MeSH
• kosti a kostní tkáň abnormality   diagnostické zobrazování   MeSH
• laminin genetika   metabolismus   MeSH
• lidé MeSH
• mutace * MeSH
• mutační analýza DNA MeSH
• signální dráha Wnt MeSH
• signální transdukce * MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• vývojové onemocnění kostí diagnóza   etiologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Mutations in genes affecting primary cilia cause ciliopathies, a diverse group of disorders often affecting skeletal development. This includes Jeune syndrome or asphyxiating thoracic dystrophy (ATD), an autosomal recessive skeletal disorder. Unraveling the responsible molecular pathology helps illuminate mechanisms responsible for functional primary cilia. We identified two families with ATD caused by loss-of-function mutations in the gene encoding adrenergic receptor kinase 1 (ADRBK1 or GRK2). GRK2 cells from an affected individual homozygous for the p.R158* mutation resulted in loss of GRK2, and disrupted chondrocyte growth and differentiation in the cartilage growth plate. GRK2 null cells displayed normal cilia morphology, yet loss of GRK2 compromised cilia-based signaling of Hedgehog (Hh) pathway. Canonical Wnt signaling was also impaired, manifested as a failure to respond to Wnt ligand due to impaired phosphorylation of the Wnt co-receptor LRP6. We have identified GRK2 as an essential regulator of skeletogenesis and demonstrate how both Hh and Wnt signaling mechanistically contribute to skeletal ciliopathies.

• MeSH
• Ellisův-van Creveldův syndrom * MeSH
• kinasa 2 receptorů spřažených s G-proteiny genetika   MeSH
• lidé MeSH
• mutace MeSH
• proteiny hedgehog * genetika   MeSH
• signální dráha Wnt MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Aplikace masivního paralelního sekvenování v oblasti onemocnění skeletu urychlila objevy genetických defektů u mnoha těchto nemocí. To také vedlo k vymezení nových klinických jednotek a identifikaci genů a drah, které dosud se skeletálními nemocemi nebyly spojovány. Tento rychlý pokrok podnítil Nosologický výbor Mezinárodní společnosti pro skeletální dysplazie revidovat a aktualizovat poslední (2015) verzi Nosologie a klasifikace genetických onemocnění skeletu. Tato nejnovější a desátá verze Nosologie zahrnuje 461 různých nemocí, které jsou klasifikovány do 42 skupin na základě jejich klinických, radiologických a/nebo molekulárních fenotypů. Je pozoruhodné, že patogenní varianty ve 437 různých genech byly nalezeny u425/461 (92 %) těchto nemocí. Poskytnutím referenčního seznamu jednotlivých jednotek a jejich příčinných genů by Nosologie měla pomoci klinikům dosáhnout přesné diagnózy u jejich pacientů a pomoci vědcům pokročit ve výzkumu kosterní biologie.

Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK's kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.

• MeSH
• buňky NIH 3T3 MeSH
• cilie metabolismus   MeSH
• CRISPR-Cas systémy MeSH
• fibroblastové růstové faktory metabolismus   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• modely u zvířat MeSH
• myši knockoutované MeSH
• myši MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• proteiny hedgehog metabolismus   MeSH
• proteomika MeSH
• receptor fibroblastových růstových faktorů, typ 1 metabolismus   MeSH
• receptor fibroblastových růstových faktorů, typ 3 genetika   metabolismus   MeSH
• receptor fibroblastových růstových faktorů, typ 4 metabolismus   MeSH
• receptory fibroblastových růstových faktorů genetika   metabolismus   MeSH
• signální transdukce MeSH
• simulace molekulového dockingu MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Receptor tyrosine kinases (RTKs) form multiprotein complexes that initiate and propagate intracellular signals and determine the RTK-specific signalling patterns. Unravelling the full complexity of protein interactions within the RTK-associated complexes is essential for understanding of RTK functions, yet it remains an understudied area of cell biology. We describe a comprehensive approach to characterize RTK interactome. A single tag immunoprecipitation and phosphotyrosine protein isolation followed by mass-spectrometry was used to identify proteins interacting with fibroblast growth factor receptor 3 (FGFR3). A total of 32 experiments were carried out in two different cell types and identified 66 proteins out of which only 20 (30.3%) proteins were already known FGFR interactors. Using co-immunoprecipitations, we validated FGFR3 interaction with adapter protein STAM1, transcriptional regulator SHOX2, translation elongation factor eEF1A1, serine/threonine kinases ICK, MAK and CCRK, and inositol phosphatase SHIP2. We show that unappreciated signalling mediators exist for well-studied RTKs, such as FGFR3, and may be identified via proteomic approaches described here. These approaches are easily adaptable to other RTKs, enabling identification of novel signalling mediators for majority of the known human RTKs.

• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• buňky NIH 3T3 MeSH
• cyklin-dependentní kinasy genetika   metabolismus   MeSH
• elongační faktor 1 genetika   metabolismus   MeSH
• endozomální třídící komplexy pro transport genetika   metabolismus   MeSH
• fosfatidylinositol-3,4,5-trisfosfát-5-fosfatasy genetika   metabolismus   MeSH
• fosfoproteiny genetika   metabolismus   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• lidé MeSH
• mapování interakce mezi proteiny MeSH
• myši MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• proteomika metody   MeSH
• receptor fibroblastových růstových faktorů, typ 3 genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• signální transdukce genetika   MeSH
• stanovení celkové genové exprese MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Studies have suggested a role for the mammalian (or mechanistic) target of rapamycin (mTOR) in skeletal development and homeostasis, yet there is no evidence connecting mTOR with the key signaling pathways that regulate skeletogenesis. We identified a parathyroid hormone (PTH)/PTH-related peptide (PTHrP)-salt-inducible kinase 3 (SIK3)-mTOR signaling cascade essential for skeletogenesis. While investigating a new skeletal dysplasia caused by a homozygous mutation in the catalytic domain of SIK3, we observed decreased activity of mTOR complex 1 (mTORC1) and mTORC2 due to accumulation of DEPTOR, a negative regulator of both mTOR complexes. This SIK3 syndrome shared skeletal features with Jansen metaphyseal chondrodysplasia (JMC), a disorder caused by constitutive activation of the PTH/PTHrP receptor. JMC-derived chondrocytes showed reduced SIK3 activity, elevated DEPTOR, and decreased mTORC1 and mTORC2 activity, indicating a common mechanism of disease. The data demonstrate that SIK3 is an essential positive regulator of mTOR signaling that functions by triggering DEPTOR degradation in response to PTH/PTHrP signaling during skeletogenesis.

• MeSH
• HEK293 buňky MeSH
• homozygot MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• lidé MeSH
• mechanistické cílové místo rapamycinového komplexu 1 metabolismus   MeSH
• mechanistické cílové místo rapamycinového komplexu 2 metabolismus   MeSH
• missense mutace genetika   MeSH
• mutantní proteiny chemie   metabolismus   MeSH
• osteogeneze * MeSH
• parathormon metabolismus   MeSH
• protein podobný parathormonu metabolismus   MeSH
• proteinkinasy chemie   nedostatek   genetika   metabolismus   MeSH
• proteolýza MeSH
• růstová ploténka metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální transdukce * MeSH
• TOR serin-threoninkinasy metabolismus   MeSH
• typy dědičnosti genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH