Long-distance signaling in nodulation directed by a CLAVATA1-like receptor kinase

Proliferation of legume nodule primordia is controlled by shoot-root signaling known as autoregulation of nodulation (AON). Mutants defective in AON show supernodulation and increased numbers of lateral roots. Here, we demonstrate that AON in soybean is controlled by the receptor-like protein kinase GmNARK (Glycine max nodule autoregulation receptor kinase), similar to Arabidopsis CLAVATA1 (CLV1). Whereas CLV1 functions in a protein complex controlling stem cell proliferation by short-distance signaling in shoot apices, GmNARK expression in the leaf has a major role in long-distance communication with nodule and lateral root primordia.

Toll-like Receptor 4 Activation Promotes Cardiac Arrhythmias By Decreasing The Transient Outward Potassium Current (ito) Through An Irf3-dependent And Myd88-independent Pathway.

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll-like receptors (TLRs) seem to be involved in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias, and the signaling pathway involved in these effects. Membrane potential was recorded in Wistar rat ventricle. Ca(2+) transients, as well as the L-type Ca(2+) current (ICaL) and the transient outward K(+) current (Ito), were recorded in isolated myocytes after 24 h exposure to the TLR4 agonist, lipopolysaccharide (LPS, 1 μg/ml). TLR4 stimulation in vitro promoted a cardiac electrical remodeling that leads to action potential prolongation associated with arrhythmic events, such as delayed afterdepolarization and triggered activity. After 24 h LPS incubation, Ito amplitude, as well as Kv4.3 and KChIP2 mRNA levels were reduced. The Ito decrease by LPS was prevented by inhibition of interferon regulatory factor 3 (IRF3), but not by inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4) or nuclear factor kappa B (NF-κB). Extrasystolic activity was present in 25% of the cells, but apart from that, Ca(2+) transients and ICaL were not affected by LPS; however, Na(+)/Ca(2+) exchanger (NCX) activity was apparently increased. We conclude that TLR4 activation decreased Ito, which increased AP duration via a MyD88-independent, IRF3-dependent pathway. The longer action potential, associated with enhanced Ca(2+) efflux via NCX, could explain the presence of arrhythmias in the LPS group.

Angiopoietin-1 and angiopoietin-2 share the same binding domains in the Tie-2 receptor involving the first Ig-like loop and the epidermal growth factor-like repeats

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) have been identified as ligands with different effector functions of the vascular assembly and maturation-mediating receptor tyrosine kinase Tie-2. To understand the molecular interactions of the angiopoietins with their receptor, we have studied the binding of Ang-1 and Ang-2 to the Tie-2 receptor. Enzyme-linked immunosorbent assay-based competition assays and co-immunoprecipitation experiments analyzing the binding of Ang-1 and Ang-2 to truncation mutants of the extracellular domain of Tie-2 showed that the first Ig-like loop of Tie-2 in combination with the epidermal growth factor (EGF)-like repeats (amino acids 1-360) is required for angiopoietin binding. The first Ig-like domain or the EGF-like repeats alone are not capable of binding Ang-1 and Ang-2. Concomitantly, we made the surprising finding that Tie-2 exon-2 knockout mice do express a mutated Tie-2 protein that lacks 104 amino acids of the first Ig-like domain. This mutant Tie-2 receptor is functionally inactive as shown by the lack of ligand binding and receptor phosphorylation. Collectively, the data show that the first 104 amino acids of the Tie-2 receptor are essential but not sufficient for angiopoietin binding. Conversely, the first 360 amino acids (Ig-like domain plus EGF-like repeats) of the Tie-2 receptor are necessary and sufficient to bind both Ang-1 and Ang-2, which suggests that differential receptor binding is not likely to be responsible for the different functions of Ang-1 and Ang-2.

Analyses of pp60$\sp{{\rm c}-src}$ and epidermal growth factor receptor protein tyrosine kinases in a human colon adenocarcinoma cell line following induction of goblet cell-like characteristics by tumor necrosis factor-alpha

Regulation of colonic epithelial cell proliferation and differentiation remains poorly understood due to the inability to design a model system which recapitulates these processes. Currently, properties of "differentiation" are studied in colon adenocarcinoma cell lines which can be induced to express some, but not all of the phenotypes of normal cells. In this thesis, the DiFi human colon adenocarcinoma cell line is utilized as an in vitro model system in which to study mucin production. In response to treatment with tumor necrosis factor-alpha, DiFi cells acquire some properties of mucin-producing goblet cells including altered morphology, increased reactivity to wheat germ agglutinin, and increased mucin production as determined by RNA expression as well as reactivity with the MUC-1 antibodies, HMFG-1 and SM-3. Thus, TNF-treated DiFi cells represent one of the few in vitro systems in which mucin expression can be induced.^ DiFi cells express an activated pp60$\sp{{\rm c}-src},$ as do most colon adenocarcinomas and derived cell lines, as well as an amplified epidermal growth factor (EGF) receptor. To assess potential changes in these enzymes during induction of differentiation characteristics, potential changes in the levels and activities of these enzymes were examined. For pp60$\sp{{\rm c}-src},$ no changes were observed in protein levels, specific activity of the kinase, cellular localization, or phosphorylation pattern as determined by Staphylococcus aureus V8 protease partial proteolytic mapping after induction of goblet cell-like phenotypic changes. These results suggest that pp60$\sp{{\rm c}-src}$ is regulated differentially in goblet cells than in absorptive cells, as down-modulation of pp60$\sp{{\rm c}-src}$ kinase occurs in the latter. Therefore, effects on pp60$\sp{{\rm c}-src}$ may be critical in colon regulation, and may be important in generating the various colonic epithelial cell types.^ In contrast to pp60$\sp{{\rm c}-src},$ EGF receptor tyrosine kinase activity decreased ($<$5-fold) after TNF treatment and at the time in which morphologic changes were observed. Similar decreases in tyrosine phosphorylation of EGF receptor were observed as assessed by immunoblotting with an anti-phosphotyrosine antibody. In addition, ($\sp{125}$I) -EGF cell surface binding was reduced approximately 3-fold following TNF treatment with a concomitant reduction in receptor affinity ($<$2-fold). These results suggest that modulation of EGF receptor may be important in goblet cell differentiation. In contrast, other published studies have demonstrated that increases in EGF receptor mRNA and in ($\sp{125}$I) -EGF binding accompany differentiation toward the absorptive cell phenotype. Therefore, differential regulation of both EGF receptor and pp60$\sp{{\rm c}-src}$ occur along the goblet cell and absorptive cell differentiation pathways. Thus, my results suggest that TNF-treated DiFi cells represent a unique system in which to study distinct patterns of regulation of pp60$\sp{{\rm c}-src}$ and EGF receptor in colonic cells, and to determine if increased MUC-1 expression is an early event in goblet cell differentiation. ^

The Role of Type I Insulin-Like Growth Factor Receptor Signaling in Breast Cancer Brain Metastasis

Brain metastasis is a common cause of mortality in cancer patients. Approximately 20-30% of breast cancer patients acquire brain metastasis, yet potential therapeutic targets remain largely unknown. The type I insulin-like growth factor receptor (IGF- IR) is known to play a role in the progression of breast cancer and is currently being investigated in the clinical setting for various types of cancer. The present study demonstrates that the IGF-IR signaling axis is constitutively active in brain-seeking sublines of breast cancer cells, driving an increase in in vitro metastatic properties. We demonstrate that IGF-IR signaling is activated in an autocrine manner as a result of IGFBP3 overexpression in brain-seeking cells. Transient and stable knockdown of IGF-IR results in a downregulation of IGF-IR downstream signaling through phospho-AKT, as well as decreased in vitro migration and invasion of MDA- MB-231Br brain-seeking cells. Using an in vivo experimental brain metastasis model, we show that IGF-IR ablation attenuates the establishment of brain metastases and prolongs survival. Finally, we demonstrate that the malignancy of brain-seeking cells is attenuated by pharmacological inhibition with picropodophyllin, an IGF-IR-specific tyrosine kinase inhibitor. Together, our data suggest that the IGF-IR is an important mediator of brain metastasis and its ablation delays the onset of brain metastases in our model system.

Control of Meristem Development by CLAVATA1 Receptor Kinase and Kinase-Associated Protein Phosphatase Interactions1

The CLAVATA1 (CLV1) gene encodes a putative receptor kinase required for the proper balance between cell proliferation and differentiation in Arabidopsis shoot and flower meristems. Impaired CLV1 signaling results in masses of undifferentiated cells at the shoot and floral meristems. Although many putative receptor kinases have been identified in plants, the mechanism of signal transduction mediated by plant receptor-like kinases is largely unknown. One potential effector of receptor kinase signaling is kinase-associated protein phosphatase (KAPP), a protein that binds to multiple plant receptor-like kinases in a phosphorylation-dependent manner. To examine a possible role for KAPP in CLV1-dependent plant development, the interaction of CLV1 and KAPP was investigated in vitro and in vivo. KAPP binds directly to autophosphorylated CLV1 in vitro and co-immunoprecipitates with CLV1 in plant extracts derived from meristematic tissue. Reduction of KAPP transcript accumulation in an intermediate clv1 mutant suppresses the mutant phenotype, and the degree of suppression is inversely correlated with KAPP mRNA levels. These data suggest that KAPP functions as a negative regulator of CLV1 signaling in plant development. This may represent a general model for the interaction of KAPP with receptor kinases.

Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach.

Advances in screening technologies allowing the identification of growth factor receptors solely by virtue of DNA or protein sequence comparison call for novel methods to isolate corresponding ligand growth factors. The EPH-like receptor tyrosine kinase (RTK) HEK (human EPH-like kinase) was identified previously as a membrane antigen on the LK63 human pre-B-cell line and overexpression in leukemic specimens and cell lines suggested a role in oncogenesis. We developed a biosensor-based approach using the immobilized HEK receptor exodomain to detect and monitor purification of the HEK ligand. A protein purification protocol, which included HEK affinity chromatography, achieved a 1.8 X 10(6)-fold purification of an approximately 23-kDa protein from human placental conditioned medium. Analysis of specific sHEK (soluble extracellular domain of HEK) ligand interactions in the first and final purification steps suggested a ligand concentration of 40 pM in the source material and a Kd of 2-3 nM. Since the purified ligand was N-terminally blocked, we generated tryptic peptides and N-terminal amino acid sequence analysis of 7 tryptic fragments of the S-pyridylethylated protein unequivocally matched the sequence for AL-1, a recently reported ligand for the related EPH-like RTK REK7 (Winslow, J.W., Moran, P., Valverde, J., Shih, A., Yuan, J.Q., Wong, S.C., Tsai, S.P., Goddard, A., Henzel, W.J., Hefti, F., Beck, K.D., & Caras, I.W. (1995) Neuron 14, 973-981). Our findings demonstrate the application of biosensor technology in ligand purification and show that AL-1, as has been found for other ligands of the EPH-like RTK family, binds more than one receptor.

A hydrogen-bonded polar network in the core of the glucagon-like peptide-1 receptor is a fulcrum for biased agonism:lessons from class B crystal structuress

The glucagon-like peptide 1 (GLP-1) receptor is a class B G protein-coupled receptor (GPCR) that is a key target for treatments for type II diabetes and obesity. This receptor, like other class B GPCRs, displays biased agonism, though the physiologic significance of this is yet to be elucidated. Previous work has implicated R2.60190 , N3.43240 , Q7.49394 , and H6.52363 as key residues involved in peptide-mediated biased agonism, with R2.60190 , N3.43240 , and Q7.49394 predicted to form a polar interaction network. In this study, we used novel insight gained from recent crystal structures of the transmembrane domains of the glucagon and corticotropin releasing factor 1 (CRF1) receptors to develop improved models of the GLP-1 receptor that predict additional key molecular interactions with these amino acids. We have introduced E6.53364 A, N3.43240 Q, Q7.49493N, and N3.43240 Q/Q7.49 Q/Q7.49493N mutations to probe the role of predicted H-bonding and charge-charge interactions in driving cAMP, calcium, or extracellular signal-regulated kinase (ERK) signaling. A polar interaction between E6.53364 and R2.60190 was predicted to be important for GLP-1- and exendin-4-, but not oxyntomodulin-mediated cAMP formation and also ERK1/2 phosphorylation. In contrast, Q7.49394 , but not R2.60190 /E6.53364 was critical for calcium mobilization for all three peptides. Mutation of N3.43240 and Q7.49394 had differential effects on individual peptides, providing evidence for molecular differences in activation transition. Collectively, this work expands our understanding of peptide-mediated signaling from the GLP-1 receptor and the key role that the central polar network plays in these events.

Insulin receptor has tyrosine kinase activity toward Shc in rat liver

Insulin induces tyrosine phosphorylation of Shc in cell cultures and in insulin-sensitive tissues of the intact rat. However, the ability of insulin receptor (IR) tyrosine kinase to phosphorylate Shc has not been previously demonstrated. In the present study, we investigated insulin-induced IR tyrosine kinase activity towards Shc. Insulin receptor was immunoprecipitated from liver extracts, before and after a very low dose of insulin into the portal vein, and incubated with immunopurified Shc from liver of untreated rats. The kinase assay was performed in vitro in the presence of exogenous ATP and the phosphorylation level was quantified by immunoblotting with antiphosphotyrosine antibody. The results demonstrate that Shc interacted with insulin receptor after infusion of insulin, and, more important, there was insulin receptor kinase activity towards immunopurified Shc. The description of this pathway in animal tissue may have an important role in insulin receptor tyrosine kinase activity toward mitogenic transduction pathways.

Efeitos do estresse hipo e hiperosmótico sobre as características do receptor à insulina e sobre a captação de glicose em branquias do caranguejo Chasmagnathus granulata

Neste trabalho investigou-se as características do receptor à insulina e a capacidade de captação de glicose nas brânquias do caranguejo Chasmagnathus granulata aclimatado a diferentes tempos (24, 72 e 144 horas) de estresse hiper e hiposmótico. Primeiramente, o cDNA do receptor para insulina foi parcialmente clonado e seqüenciado em brânquias posteriores de Chasmagnathus granulata. A seqüência peptídica mostrou a presença de 39 aminoácidos e foi designada CGIRLTK (C. granulata insulina receptor-like tyrosine kinase). Esta seqüência apresentou significativa homologia com o domínio tirosina quinase da subunidade b dos receptores para insulina de mamíferos (69%) e de Drosophila (74%). Sítios de ligação à insulina foram caracterizados nas membranas plasmáticas das brânquias através do estudo de ligação com 125I-insulina. A atividade tirosina quinase foi determinada pela capacidade do CGIRLTK de fosforilar o substrato sintético poly (Glu; Tyr 4:1). A captação de glicose foi avaliada pela captação de [14C] 2-deoxi-D-glicose pelo tecido branquial. Nas brânquias posteriores a insulina bovina estimulou significativamente a fosforilação do CGIRLTK nos animais aclimatados a 20‰ de salinidade (controle), já nas brânquias anteriores este estímulo não foi observado. O estresse hiperosmótico (34 ‰ de salinidade) levou a uma diminuição do número e da afinidade dos receptores à insulina nas brânquias posteriores, bem como a uma redução na atividade tirosina quinase. A captação de glicose não mudou durante os tempos de estresse osmótico estudados Esses resultados mostram que o estresse hiperosmótico modifica a sinalização da insulina, causando um estado de resistência à insulina nas brânquias posteriores. Nenhuma mudança foi observada na concentração dos receptores à insulina nas brânquias posteriores de caranguejos aclimatados durante 24 horas ao estresse hiposmótico (0‰). Contudo, foi observada uma redução na afinidade dos receptores pela insulina bovina. A fosforilação do CGIRLTK diminui às 24 horas de estresse e retornou aos valores basais às 144horas. A captação de glicose não foi alterada significativamente. Os resultados sugerem que o estresse hiposmótico modifica as características do CGIRLTK nas brânquias posteriores de C. granulata de forma tempo-dependente. Essas mudanças são parte dos ajustes necessários à sobrevivência à baixa salinidade. Nas brânquias anteriores, durante aclimatação ao estresse hiperosmótico, foi observada redução da concentração e da capacidade de fosforilação dos receptores insulínicos. Contudo, a insulina bovina não estimulou a fosforilação nas brânquias anteriores durante o estresse Nenhuma alteração foi observada na concentração e na afinidade de receptores à insulina nas brânquias anteriores após 24 horas de estresse hiposmótico. A fosforilação do receptor à insulina diminuiu após 24 horas de estresse e voltou aos valores basais após 72 horas. A capacidade de captação de glicose, por sua vez, não foi modificada em função de mudanças na osmoliridade do ambiente. Assim como no estresse hiperosmótico, a insulina bovina não estimulou a fosforilação nas brânquias anteriores no estresse hiposmótico. Os resultados deste trabalho demonstram que o estresse osmótico modifica as características do CGILRTK e conseqüentemente a transdução do sinal insulínico nas brânquias. As respostas às alterações de salinidade dependem do tipo de estresse ao qual o animal é submetido e da brânquia estudada (anterior ou posterior). As mudanças observadas no sinal insulínico fazem parte dos ajustes necessários para a regulação osmótica frente às mudanças ambientais de salinidade.

The FGFRL1 receptor is shed from cell membranes, binds fibroblast growth factors (FGFs), and antagonizes FGF signaling in Xenopus embryos

FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs.

Biology of FGFRL1, the fifth fibroblast growth factor receptor

FGFRL1 (fibroblast growth factor receptor like 1) is the most recently discovered member of the FGFR family. It contains three extracellular Ig-like domains similar to the classical FGFRs, but it lacks the protein tyrosine kinase domain and instead contains a short intracellular tail with a peculiar histidine-rich motif. The gene for FGFRL1 is found in all metazoans from sea anemone to mammals. FGFRL1 binds to FGF ligands and heparin with high affinity. It exerts a negative effect on cell proliferation, but a positive effect on cell differentiation. Mice with a targeted deletion of the Fgfrl1 gene die perinatally due to alterations in their diaphragm. These mice also show bilateral kidney agenesis, suggesting an essential role for Fgfrl1 in kidney development. A human patient with a frameshift mutation exhibits craniosynostosis, arguing for an additional role of FGFRL1 during bone formation. FGFRL1 contributes to the complexity of the FGF signaling system.

Evidence that the novel receptor FGFRL1 signals indirectly via FGFR1

Fibroblast growth factor (FGF) receptor-like protein 1 (FGFRL1) is a recently discovered member of the FGF receptor (FGFR) family. Similar to the classical FGFRs, it contains three extracellular immunoglobulin-like domains and interacts with FGF ligands. However, in contrast to the classical receptors, it does not contain any intracellular tyrosine kinase domain and consequently cannot signal by transphosphorylation. In mouse kidneys, FgfrL1 is expressed primarily at embryonic stages E14-E15 in regions where nascent nephrons develop. In this study, we used whole-mount in situ hybridization to show the spatial pattern of five different Fgfrs in the developing mouse kidney. We compared the expression pattern of FgfrL1 with that of other Fgfrs. The expression pattern of FgfrL1 closely resembled that of Fgfr1, but clearly differed from that of Fgfr2‑Fgfr4. It is therefore conceivable that FgfrL1 signals indirectly via Fgfr1. The mechanisms by which FgfrL1 affects the activity of Fgfr1 remain to be elucidated.

Antiseptic solutions modulate the paracrine-like activity of bone chips: differential impact of chlorhexidine and sodium hypochlorite

AIM: Chemical decontamination increases the availability of bone grafts; however, it is unclear whether antiseptic processing changes the biological activity of bone. MATERIALS AND METHODS: Bone chips were incubated with 4 different antiseptic solutions including (1) povidone-iodine (0.5%), (2) chlorhexidine diguluconate (0.2%), (3) hydrogen peroxide (1%) and (4) sodium hypochlorite (0.25%). After 10 minutes of incubation, changes in the capacity of the bone-conditioned medium to modulate gene expression of gingival fibroblasts was investigated. RESULTS: Conditioned medium obtained from freshly prepared bone chips increased the expression of TGF-β target genes interleukin 11 (IL11), proteoglycan4 (PRG4), NADPH oxidase 4 (NOX4), and decreased the expression of adrenomedullin (ADM), and pentraxin 3 (PTX3) in gingival fibroblasts. Incubation of bone chips with 0.2% chlorhexidine, followed by vigorously washing resulted in a bone-conditioned medium with even higher expression of IL11, PRG4, and NOX4. These findings were also found with a decrease in cell viability and an activation of apoptosis signaling. Chlorhexidine alone, at low concentrations, increased IL11, PRG4 and NOX4 expression, independent of the TGF-β receptor I kinase activity. In contrast, 0.25% sodium hypochlorite almost entirely abolished the activity of bone-conditioned medium, while the other two antiseptic solutions, 1% hydrogen peroxide and 0.5% povidone-iodine, had relatively no impact, respectively. CONCLUSION: These in vitro findings demonstrate that incubation of bone chips with chlorhexidine differentially affects the activity of the respective bone-conditioned medium compared to the other antiseptic solutions. The data further suggest that the main effects are caused by chlorhexidine remaining in the bone-conditioned medium after repeated washing of the bone chips. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. KEYWORDS: Autografts; TGF-β; antiseptic solution; bone; bone conditioned medium; bone supernatant; chlorhexidine; hydrogen peroxide; povidone-iodine; sodium hypochlorite

Transforming growth factor beta signaling is essential for the autonomous formation of cartilage-like tissue by expanded chondrocytes.

Cartilage is a tissue with limited self-healing potential. Hence, cartilage defects require surgical attention to prevent or postpone the development of osteoarthritis. For cell-based cartilage repair strategies, in particular autologous chondrocyte implantation, articular chondrocytes are isolated from cartilage and expanded in vitro to increase the number of cells required for therapy. During expansion, the cells lose the competence to autonomously form a cartilage-like tissue, that is in the absence of exogenously added chondrogenic growth factors, such as TGF-βs. We hypothesized that signaling elicited by autocrine and/or paracrine TGF-β is essential for the formation of cartilage-like tissue and that alterations within the TGF-β signaling pathway during expansion interfere with this process. Primary bovine articular chondrocytes were harvested and expanded in monolayer culture up to passage six and the formation of cartilage tissue was investigated in high density pellet cultures grown for three weeks. Chondrocytes expanded for up to three passages maintained the potential for autonomous cartilage-like tissue formation. After three passages, however, exogenous TGF-β1 was required to induce the formation of cartilage-like tissue. When TGF-β signaling was blocked by inhibiting the TGF-β receptor 1 kinase, the autonomous formation of cartilage-like tissue was abrogated. At the initiation of pellet culture, chondrocytes from passage three and later showed levels of transcripts coding for TGF-β receptors 1 and 2 and TGF-β2 to be three-, five- and five-fold decreased, respectively, as compared to primary chondrocytes. In conclusion, the autonomous formation of cartilage-like tissue by expanded chondrocytes is dependent on signaling induced by autocrine and/or paracrine TGF-β. We propose that a decrease in the expression of the chondrogenic growth factor TGF-β2 and of the TGF-β receptors in expanded chondrocytes accounts for a decrease in the activity of the TGF-β signaling pathway and hence for the loss of the potential for autonomous cartilage-like tissue formation.