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
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
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
Cilia project from almost every cell integrating extracellular cues with signaling pathways. Constitutive activation of FGFR3 signaling produces the skeletal disorders achondroplasia (ACH) and thanatophoric dysplasia (TD), but many of the molecular mechanisms underlying these phenotypes remain unresolved. Here, we report in vivo evidence for significantly shortened primary cilia in ACH and TD cartilage growth plates. Using in vivo and in vitro methodologies, our data demonstrate that transient versus sustained activation of FGF signaling correlated with different cilia consequences. Transient FGF pathway activation elongated cilia, while sustained activity shortened cilia. FGF signaling extended primary cilia via ERK MAP kinase and mTORC2 signaling, but not through mTORC1. Employing a GFP-tagged IFT20 construct to measure intraflagellar (IFT) speed in cilia, we showed that FGF signaling affected IFT velocities, as well as modulating cilia-based Hedgehog signaling. Our data integrate primary cilia into canonical FGF signal transduction and uncover a FGF-cilia pathway that needs consideration when elucidating the mechanisms of physiological and pathological FGFR function, or in the development of FGFR therapeutics.
- MeSH
- achondroplazie genetika patofyziologie MeSH
- buňky NIH 3T3 MeSH
- chondrocyty metabolismus MeSH
- chrupavka metabolismus MeSH
- cilie patologie fyziologie MeSH
- ciliopatie genetika patofyziologie MeSH
- fenotyp MeSH
- fibroblastové růstové faktory metabolismus MeSH
- lidé MeSH
- myši MeSH
- primární buněčná kultura MeSH
- receptor fibroblastových růstových faktorů, typ 3 genetika metabolismus MeSH
- růstová ploténka metabolismus MeSH
- signální transdukce fyziologie MeSH
- thanatoforní dysplazie genetika patofyziologie 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
Lysine hydroxylation of type I collagen telopeptides varies from tissue to tissue, and these distinct hydroxylation patterns modulate collagen cross-linking to generate a unique extracellular matrix. Abnormalities in these patterns contribute to pathologies that include osteogenesis imperfecta (OI), fibrosis, and cancer. Telopeptide procollagen modifications are carried out by lysyl hydroxylase 2 (LH2); however, little is known regarding how this enzyme regulates hydroxylation patterns. We identified an ER complex of resident chaperones that includes HSP47, FKBP65, and BiP regulating the activity of LH2. Our findings show that FKBP65 and HSP47 modulate the activity of LH2 to either favor or repress its activity. BiP was also identified as a member of the complex, playing a role in enhancing the formation of the complex. This newly identified ER chaperone complex contributes to our understanding of how LH2 regulates lysyl hydroxylation of type I collagen C-telopeptides to affect the quality of connective tissues. © 2017 American Society for Bone and Mineral Research.
- MeSH
- biologické modely MeSH
- buněčné linie MeSH
- hmotnostní spektrometrie MeSH
- hydroxylace MeSH
- kolagen typu I metabolismus MeSH
- lidé MeSH
- lysin metabolismus MeSH
- lysinhydroxylasa metabolismus MeSH
- multiproteinové komplexy metabolismus MeSH
- mutace genetika MeSH
- myši MeSH
- peptidy metabolismus MeSH
- povrchová plasmonová rezonance MeSH
- prokolagen metabolismus MeSH
- proteiny tepelného šoku HSP47 metabolismus MeSH
- proteiny tepelného šoku metabolismus MeSH
- proteiny vázající takrolimus metabolismus MeSH
- stabilita enzymů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The short rib polydactyly syndromes (SRPS) are a group of recessively inherited, perinatal-lethal skeletal disorders primarily characterized by short ribs, shortened long bones, varying types of polydactyly and concomitant visceral abnormalities. Mutations in several genes affecting cilia function cause SRPS, revealing a role for cilia function in skeletal development. To identify additional SRPS genes and discover novel ciliary molecules required for normal skeletogenesis, we performed exome sequencing in a cohort of patients and identified homozygosity for a missense mutation, p.E80K, in Intestinal Cell Kinase, ICK, in one SRPS family. The p.E80K mutation abolished serine/threonine kinase activity, resulting in altered ICK subcellular and ciliary localization, increased cilia length, aberrant cartilage growth plate structure, defective Hedgehog and altered ERK signalling. These data identify ICK as an SRPS-associated gene and reveal that abnormalities in signalling pathways contribute to defective skeletogenesis.
- MeSH
- cilie genetika patologie MeSH
- exom genetika MeSH
- kojenec MeSH
- kostra abnormality růst a vývoj MeSH
- lidé MeSH
- MAP kinasový signální systém MeSH
- mnohočetné abnormality genetika patofyziologie MeSH
- protein-serin-threoninkinasy genetika MeSH
- proteiny hedgehog genetika MeSH
- rodokmen MeSH
- sekvenční analýza DNA MeSH
- signální transdukce MeSH
- syndrom krátkého žebra a polydaktylie genetika patologie MeSH
- těhotenství MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
