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Significance A properly functioning primary cilium is prerequisite for both normal development and aging of all ciliated organisms, including humans. In vertebrates, the signaling of Hedgehog family morphogens depends entirely on primary cilium. Recently, we reported that fibroblast growth factors (FGF) signaling interacts with that of Hedgehog, and that this is a consequence of FGF regulating length of the cilium and speed of processes that happen therein. In this report, we provide a molecular mechanism of such interaction, identifying intestinal cell kinase as a mediator of the FGF-induced changes in the ciliary morphology and function. This expands our understanding how FGF signaling regulates intracellular processes, and how aberrant FGF signaling contributes to diseases, such as achondroplasia and cancer., 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.

Fibroblast growth factors (FGFs) serve numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability. Here we tackle this problem using a generalizable computer-assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function. The data from the characterization of stabilized FGF2 showed a remarkable prediction potential of in silico methods and provided insight into the unfolding mechanism of the protein. The molecule holds a considerable promise for stem cell research and medical or pharmaceutical applications.

Many tyrosine kinase inhibitors (TKIs) have failed to reach human use due to insufficient activity in clinical trials. However, the failed TKIs may still benefit patients if their other kinase targets are identified by providing treatment focused on syndromes driven by these kinases. Here, we searched for novel targets of AZD1480, an inhibitor of JAK2 kinase that recently failed phase two cancer clinical trials due to a lack of activity. Twenty seven human receptor tyrosine kinases (RTKs) and 153 of their disease-associated mutants were in-cell profiled for activity in the presence of AZD1480 using a newly developed RTK plasmid library. We demonstrate that AZD1480 inhibits ALK, LTK, FGFR1-3, RET and TRKA-C kinases and uncover a physical basis of this specificity. The RTK activity profiling described here facilitates inhibitor repurposing by enabling rapid and efficient identification of novel TKI targets in cells.

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., This study identifies GRK2 as a regulator of human skeletogenesis. Loss of GRK2 deregulates the function of two major morphogens in the bone ‐ Hedgehog and canonical Wnt signaling, and manifests in autosomal recessive skeletal ciliopathy syndrome, asphyxiating thoracic dystrophy or Jeune syndrome.

The blocking of specific protein-protein interactions using nanoparticles is an emerging alternative to small molecule-based therapeutic interventions. However, the nanoparticles designed as "artificial proteins" generally require modification of their surface with (bio)organic molecules and/or polymers to ensure their selectivity and specificity of action. Here, we show that nanosized diamond crystals (nanodiamonds, NDs) without any synthetically installed (bio)organic interface enable the specific and efficient targeting of the family of extracellular signalling molecules known as fibroblast growth factors (FGFs). We found that low nanomolar solutions of detonation NDs with positive-potential strongly associate with multiple FGF ligands present at sub-nanomolar concentrations and effectively neutralize the effects of FGF signalling in cells without interfering with other growth factor systems and serum proteins unrelated to FGFs. We identified an evolutionarily conserved FGF recognition motif, 17 amino acids long, that contributes to the selectivity of the ND-FGF interaction. In addition, we inserted this motif into a de novo constructed chimeric protein, which significantly improved its interaction with NDs. We demonstrated that the interaction of NDs, as purely inorganic nanoparticles, with proteins can mitigate pathological FGF signalling and promote the restoration of cartilage growth in a mouse limb explant model. Based on our observations, we foresee that NDs may potentially be applied as nano therapeutics to neutralize disease-related activities of FGFs in vivo. (C) 2018 The Authors. Published by Elsevier Ltd.

Sustained activation of extracellular signal-regulated kinase (ERK) drives pathologies caused by mutations in fibroblast growth factor receptors (FGFRs). We previously identified the inositol phosphatase SHIP2 (also known as INPPL1) as an FGFR-interacting protein and a target of the tyrosine kinase activities of FGFR1, FGFR3, and FGFR4. We report that loss of SHIP2 converted FGF-mediated sustained ERK activation into a transient signal and rescued cell phenotypes triggered by pathologic FGFR-ERK signaling. Mutant forms of SHIP2 lacking phosphoinositide phosphatase activity still associated with FGFRs and did not prevent FGF-induced sustained ERK activation, demonstrating that the adaptor rather than the catalytic activity of SHIP2 was required. SHIP2 recruited Src family kinases to the FGFRs, which promoted FGFR-mediated phosphorylation and assembly of protein complexes that relayed signaling to ERK. SHIP2 interacted with FGFRs, was phosphorylated by active FGFRs, and promoted FGFR-ERK signaling at the level of phosphorylation of the adaptor FRS2 and recruitment of the tyrosine phosphatase PTPN11. Thus, SHIP2 is an essential component of canonical FGF-FGFR signal transduction and a potential therapeutic target in FGFR-related disorders.

ObjectiveMutations of the fibroblast growth factor receptor 3 (FGFR3) cause various forms of short stature, of which the least severe phenotype is hypochondroplasia, mainly characterized by disproportionate short stature. Testing for an FGFR3 mutation is currently not part of routine diagnostic testing in children with short stature without disproportion.DesignA three-generation family A with dominantly transmitted proportionate short stature was studied by whole-exome sequencing to identify the causal gene mutation. Functional studies and protein modeling studies were performed to confirm the pathogenicity of the mutation found in FGFR3. We performed Sanger sequencing in a second family B with dominant proportionate short stature and identified a rare variant in FGFR3.MethodsExome sequencing and/or Sanger sequencing was performed, followed by functional studies using transfection of the mutant FGFR3 into cultured cells; homology modeling was used to construct a three-dimensional model of the two FGFR3 variants.ResultsA novel p.M528I mutation in FGFR3 was detected in family A, which segregates with short stature and proved to be activating in vitro. In family B, a rare variant (p.F384L) was found in FGFR3, which did not segregate with short stature and showed normal functionality in vitro compared with WT.ConclusionsProportionate short stature can be caused by a mutation in FGFR3. Sequencing of this gene can be considered in patients with short stature, especially when there is an autosomal dominant pattern of inheritance. However, functional studies and segregation studies should be performed before concluding that a variant is pathogenic.

Aberrant fibroblast growth factor (FGF) signaling disturbs chondrocyte differentiation in skeletal dysplasia, but the mechanisms underlying this process remain unclear. Recently, FGF was found to activate canonical WNT/β-catenin pathway in chondrocytes via Erk MAP kinase-mediated phosphorylation of WNT co-receptor Lrp6. Here, we explore the cellular consequences of such a signaling interaction. WNT enhanced the FGF-mediated suppression of chondrocyte differentiation in mouse limb bud micromass and limb organ cultures, leading to inhibition of cartilage nodule formation in micromass cultures, and suppression of growth in cultured limbs. Simultaneous activation of the FGF and WNT/β-catenin pathways resulted in loss of chondrocyte extracellular matrix, expression of genes typical for mineralized tissues and alteration of cellular shape. WNT enhanced the FGF-mediated downregulation of chondrocyte proteoglycan and collagen extracellular matrix via inhibition of matrix synthesis and induction of proteinases involved in matrix degradation. Expression of genes regulating RhoA GTPase pathway was induced by FGF in cooperation with WNT, and inhibition of the RhoA signaling rescued the FGF/WNT-mediated changes in chondrocyte cellular shape. Our results suggest that aberrant FGF signaling cooperates with WNT/β-catenin in suppression of chondrocyte differentiation.

Fibroblast growth factors (FGFs) play numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability in vivo and in vitro. Here we tackle this problem using a generalizable computer-assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function.

Summary Objective Statins are widely used drugs for cholesterol lowering, which were recently found to counteract the effects of aberrant fibroblast growth factor receptor (FGFR3) signaling in cell and animal models of FGFR3-related chondrodysplasia. This opened an intriguing therapeutic possibility for human dwarfing conditions caused by gain-of-function mutations in FGFR3, although the mechanism of statin action on FGFR3 remains unclear. Here, we determine the effect of statins on FGFR signaling in chondrocytes. Design Cultured chondrocyte cell lines, mouse embryonic tibia cultures and limb bud micromasses were treated with FGF2 to activate FGFR signaling. The effects of atorvastatin, fluvastatin, lovastatin and pravastatin on FGFR3 protein stability and on FGFR-mediated chondrocyte growth-arrest, loss of extracellular matrix (ECM), induction of premature senescence and hypertrophic differentiation were evaluated. Results Statins did not alter the level of FGFR3 protein expression nor produce any effect on FGFR-mediated inhibition of chondrocyte proliferation and hypertrophic differentiation in cultured chondrocyte cell lines, mouse tibia cultures or limb bud micromasses. Conclusion We conclude that statins do not inhibit the FGFR signaling in chondrocytes. Therefore the statin-mediated rescue of FGFR3-related chondrodysplasia, described before, is likely not intrinsic to the growth plate cartilage.

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.

Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) cause the most common genetic form of human dwarfism, achondroplasia (ACH). Small chemical inhibitors of FGFR tyrosine kinase activity are considered to be viable option for treating ACH, but little experimental evidence supports this claim. We evaluated five FGFR tyrosine kinase inhibitors (TKIs) (SU5402, PD173074, AZD1480, AZD4547 and BGJ398) for their activity against FGFR signaling in chondrocytes. All five TKIs strongly inhibited FGFR activation in cultured chondrocytes and limb rudiment cultures, completely relieving FGFR-mediated inhibition of chondrocyte proliferation and maturation. In contrast, TKI treatment of newborn mice did not improve skeletal growth and had lethal toxic effects on the liver, lungs and kidneys. In cell-free kinase assays as well as in vitro and in vivo cell assays, none of the tested TKIs demonstrated selectivity for FGFR3 over three other FGFR tyrosine kinases. In addition, the TKIs exhibited significant off-target activity when screened against a panel of 14 unrelated tyrosine kinases. This was most extensive in SU5402 and AZD1480, which inhibited DDR2, IGF1R, FLT3, TRKA, FLT4, ABL and JAK3 with efficiencies similar to or greater than those for FGFR. Low target specificity and toxicity of FGFR TKIs thus compromise their use for treatment of ACH. Conceptually, different avenues of therapeutic FGFR3 targeting should be investigated.

Fibroblast growth factor (FGF) signalling appears essential for the regulation of limb development, but a full complexity of this regulation remains unclear. Here, we addressed the effect of three different chemical inhibitors of FGF receptor tyrosine kinases (FGFR) on growth and patterning of the chicken wings. The inhibitor PD173074 caused shorter and thinner wing when using lower concentration. Microinjection of higher PD173074 concentrations (25 and 50 mmol/L) into the wing bud at stage 20 resulted in the development of small wing rudiment or the total absence of the wing. Skeletal analysis revealed the absence of the radius but not ulna, deformation of metacarpal bones and/or a reduction of digits. Treatment with PD161570 resembled the effects of PD173074. NF449 induced shortening and deformation of the developing wing with reduced autopodium. These malformed embryos mostly died at the stage HH25-29. PD173074 reduced chondrogenesis also in the limb micromass cultures together with early inhibition of cartilaginous nodule formation, evidenced by lack of sulphated proteoglycan and peanut agglutinin expression. The effect of FGFR inhibition on limb development observed here was unlikely mediated by excessive cell death as none of the inhibitors caused massive apoptosis at low concentrations. More probably, FGFR inhibition decreased both the proliferation and adhesion of mesenchymal chondroprogenitors. We conclude that FGFR signalling contributes to the regulation of the anterior-posterior patterning of zeugopod during chicken limb development.

The inevitable accumulation of chromosomal abnormalities in human embryonic stem cells (hESCs) during in vitro expansion represents a considerable obstacle for cell replacement therapies. To determine the source of chromosomal abnormalities, we examined hESCs maintained in culture for over 55 months for defects in telomere maintenance and DNA repair. Although prolonged culture affected neither telomerase activity nor nonhomologous end joining, the efficiency of base excision repair (BER) was significantly decreased and correlated with reduced expression of apurinic/apyrimidinic endonuclease 1 (APE1), the major nuclease required for BER. Interestingly, the expression of other BER enzymes was unchanged. Addition of human recombinant APE1 protein to nuclear extracts from late passage hESCs increased BER efficiency to the level typical of early passage hESCs. The link between BER and double-strand breaks (DSB) was demonstrated by decreased DSB release after downregulation of APE1 in early passage hESCs via siRNA. Correspondingly lower APE1 level in late passage hESC resulted in slower and less intensive but long lasting DSB release upon ionizing radiation (IR). Downregulation of APE1 in early passage hESCs also led to approximately 30% decrease in γ-H2AX signaling following IR, similar to that in late passage hESCs. We suggest that downregulation of APE1 significantly contributes to the failure of BER during long-term culture of hESCs, and further that BER failure is one of the factors affecting the genomic instability of hESCs by altering BER-dependent DSB release and cell cycle/checkpoint signaling.