The FGF signaling pathway plays an important role in the regulation of limb development, controlling cell migration, proliferation, differentiation, and apoptosis. Sprouty proteins act as antagonists of the FGF pathway and control the extent of FGF signaling as part of a negative feedback loop. Sprouty2/4 deficient mice evince defects in endochondral bone formation and digit patterning in their forelimbs, with pathogenesis recently related to ciliopathies. To understand the mechanisms behind these pathologies, the limb defects in Sprouty2+/-;Sprouty4-/- male and female mice were characterized and correlated to the dynamic expression patterns of Sprouty2 and Sprouty4, and the impact on the main signaling centers of the limb bud was assessed. Sprouty2 and Sprouty4 exhibited dynamic expressions during limb development. Interestingly, despite similar expression patterns in all limbs, the hindlimbs did not evince any obvious alterations in development, while the forelimbs showed consistent phenotypes of variable severity. Prenatally as well as postnatally, the left forelimb was significantly more severely affected than the right one. A broad variety of pathologies was present in the autopodium of the forelimb, including changes in digit number, size, shape, and number of bones, hand clefts, and digit fusions. Ectopic ossification of bones and abnormal bone fusions detected in micro-CT scans were frequently observed in the digital as well as in the carpal and metacarpal areas. Sprouty2+/-;Sprouty4-/- limb buds showed patchy loss of Fgf8 expression in the apical ectodermal ridge, and a loss of tissue underlying these regions. The zone of polarizing activity was also impacted, with lineage analysis highlighting a change in the contribution of Sonic hedgehog expressing cells. These findings support the link between Sproutys and Hedgehog signaling during limb development and highlight the importance of Sprouty2 and Sprouty4 in controlling early signaling centers in the limb.

• Klíčová slova
• FGF signaling, RTKs, Shh, apical ectodermal ridge, autopodium pathologies, ciliopathy, genetic animal models, limb patterning, micro-CT, zone of polarizing activity,
• Publikační typ
• časopisecké články MeSH

Achondroplasia is the most common form of human dwarfism caused by mutations in the FGFR3 receptor tyrosine kinase. Current therapy begins at 2 years of age and improves longitudinal growth but does not address the cranial malformations including midface hypoplasia and foramen magnum stenosis, which lead to significant otolaryngeal and neurologic compromise. A recent clinical trial found partial restoration of cranial defects with therapy starting at 3 months of age, but results are still inconclusive. The benefits of achondroplasia therapy are therefore controversial, increasing skepticism among the medical community and patients. We used a mouse model of achondroplasia to test treatment protocols aligned with human studies. Early postnatal treatment (from day 1) was compared with late postnatal treatment (from day 4, equivalent to ~5 months in humans). Animals were treated with the FGFR3 inhibitor infigratinib and the effect on skeleton was thoroughly examined. We show that premature fusion of the skull base synchondroses occurs immediately after birth and leads to defective cranial development and foramen magnum stenosis in the mouse model to achondroplasia. This phenotype appears significantly restored by early infigratinib administration when compared with late treatment, which provides weak to no rescue. In contrast, the long bone growth is similarly improved by both early and late protocols. We provide clear evidence that immediate postnatal therapy is critical for normalization of skeletal growth in both the cranial base and long bones and the prevention of sequelae associated with achondroplasia. We also describe the limitations of early postnatal therapy, providing a paradigm-shifting argument for the development of prenatal therapy for achondroplasia.

The article provides clear evidence that achondroplasia should be treated immediately after birth, not only to increase height (appendicular growth), but more importantly to prevent defective cranial skeletogenesis and associated severe neurological complications. Although later treatment promotes growth of the long bones (achondroplasia patients grow taller), the defective head skeleton that forms before and/or early after birth cannot be restored if therapy is not started immediately after birth. We also describe the limitations of postnatal treatment and make a strong case for the development of prenatal therapy for achondroplasia, which appears necessary for a comprehensive treatment of this condition.

• Klíčová slova
• Fgfr3, achondroplasia, fibroblast growth factor, infigratinib, postnatal, treatment,
• MeSH
• achondroplazie * patologie   farmakoterapie   MeSH
• lebka patologie   účinky léků   MeSH
• lidé MeSH
• modely nemocí na zvířatech * MeSH
• myši MeSH
• receptor fibroblastových růstových faktorů, typ 3 * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• receptor fibroblastových růstových faktorů, typ 3 * MeSH

Ciliopathies represent a disease class characterized by a broad range of phenotypes including polycystic kidneys and skeletal anomalies. Ciliopathic skeletal phenotypes are among the most common and most difficult to treat due to a poor understanding of the pathological mechanisms leading to disease. Using an avian model (talpid2) for a human ciliopathy with both kidney and skeletal anomalies (orofaciodigital syndrome 14), we identified disruptions in the FGF23-PTH axis that resulted in reduced calcium uptake in the developing mandible and subsequent micrognathia. Although pharmacological intervention with the U.S. Food and Drug Administration (FDA)-approved pan-FGFR inhibitor AZD4547 alone rescued expression of the FGF target SPRY2, it did not significantly rescue micrognathia. In contrast, treatment with a cocktail of AZD4547 and teriparatide acetate, a PTH agonist and FDA-approved treatment for osteoporosis, resulted in molecular, cellular and phenotypic rescue of ciliopathic micrognathia in talpid2 mutants. Together, these data provide novel insight into pathological molecular mechanisms associated with ciliopathic skeletal phenotypes and a potential therapeutic strategy for a pleiotropic disease class with limited to no treatment options.

• Klíčová slova
• talpid2, C2CD3, Ciliopathies, FGF, Micrognathia, Primary cilia,
• MeSH
• cilie metabolismus   MeSH
• ciliopatie * farmakoterapie   genetika   metabolismus   MeSH
• fenotyp MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• mikrognacie * metabolismus   patologie   MeSH
• proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• intracelulární signální peptidy a proteiny MeSH
• membránové proteiny MeSH
• proteiny MeSH
• SPRY2 protein, human MeSH Prohlížeč

A single primary cilium projects from most vertebrate cells to guide cell fate decisions. A growing list of signaling molecules is found to function through cilia and control ciliogenesis, including the fibroblast growth factor receptors (FGFR). Aberrant FGFR activity produces abnormal cilia with deregulated signaling, which contributes to pathogenesis of the FGFR-mediated genetic disorders. FGFR lesions are also found in cancer, raising a possibility of cilia involvement in the neoplastic transformation and tumor progression. Here, we focus on FGFR gene fusions, and discuss the possible mechanisms by which they function as oncogenic drivers. We show that a substantial portion of the FGFR fusion partners are proteins associated with the centrosome cycle, including organization of the mitotic spindle and ciliogenesis. The functions of centrosome proteins are often lost with the gene fusion, leading to haploinsufficiency that induces cilia loss and deregulated cell division. We speculate that this complements the ectopic FGFR activity and drives the FGFR fusion cancers.

• Klíčová slova
• FGFR, FGFR fusion, cancer, centrosome, centrosome cycle, cilia, fibroblast growth factor receptor, neoplastic transformation, oncogenic driver, primary cilia,
• MeSH
• centrozom metabolismus   MeSH
• cilie * metabolismus   patologie   MeSH
• lidé MeSH
• nádorová transformace buněk patologie   MeSH
• nádory * metabolismus   patologie   MeSH
• onkogenní fúze MeSH
• receptory fibroblastových růstových faktorů metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• receptory fibroblastových růstových faktorů MeSH

Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-β. However, gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/β-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/β-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast, second model proposes a positive role of WNT/β-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/β-catenin pathway, β-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/β-catenin pathway in cell lines (RPE-1, NIH3T3, and HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6, or AXIN1/2 we show that neither activation nor deactivation of the WNT/β-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/β-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/β-catenin pathway in some systems seems to moderately suppress ciliogenesis.

• Klíčová slova
• HEK293, NIH3T3, RPE-1, Wnt/β-catenin, Wnt3a, cell signaling, ciliogenesis, primary cilia,
• 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.

• Klíčová slova
• GRK2, Wnt, asphyxiating thoracic dystrophy, hedgehog, smoothened,
• 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
• Názvy látek
• GRK2 protein, human MeSH Prohlížeč
• kinasa 2 receptorů spřažených s G-proteiny MeSH
• proteiny hedgehog * MeSH

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.

• Klíčová slova
• FGFR, ICK, cilia length, fibroblast growth factor, intestinal cell kinase,
• 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
• Názvy látek
• CILK1 protein, human MeSH Prohlížeč
• Cilk1 protein, mouse MeSH Prohlížeč
• FGFR1 protein, human MeSH Prohlížeč
• FGFR3 protein, human MeSH Prohlížeč
• FGFR4 protein, human MeSH Prohlížeč
• fibroblastové růstové faktory MeSH
• protein-serin-threoninkinasy MeSH
• proteiny hedgehog MeSH
• receptor fibroblastových růstových faktorů, typ 1 MeSH
• receptor fibroblastových růstových faktorů, typ 3 MeSH
• receptor fibroblastových růstových faktorů, typ 4 MeSH
• receptory fibroblastových růstových faktorů MeSH