Immunolocalization of notch signaling protein molecules in a maxillary chondrosarcoma and its recurrent tumor.
"Both primary and recurrent tumors were histopathologically diagnosed as conventional hyaline chondrosarcoma (WHO Grade I). Hypercellular tumor areas strongly expressed Notch3 and Jagged1 in spindle and pleomorphic cells suggesting up-regulation of these protein molecules at sites of tumor proliferation. Expression patterns were distinct with some overlap. Differentiated malignant and atypical chondrocytes demonstrated variable expression levels of Jagged1, and weak to absent staining for Notch1, 4 and Delta1. Protein immunolocalization was largely membranous and cytoplasmic, sometimes outlining the lacunae of malignant chondrocytes. Hyaline cartilage demonstrated a diffuse or granular precipitation of Jagged1 suggesting presence of soluble Jagged1 activity at sites of abnormal chondrogenesis. No immunoreactivity for the other Notch members was observed. Calcified cartilage was consistently Notch-negative indicating down-regulation of Notch with cartilage maturation[upregulate Notch's to decrease cartilage maturation and to grow taller for longer?].
Results indicate that Notch signaling pathway may participate in cellular differentiation and proliferation in chondrosarcoma. Findings implicate Notch3 and Jagged1 as key molecules that influence the differentiation and maturation of cells of chondrogenic lineage."
"Notch receptors are structurally homologous transmembrane proteins with distinct differences in their extracellular and intracellular domains (ICD)."
"Binding of Notch ligands to their receptors on neighboring cells induce proteolytic cleavages, releasing Notch ICD which translocates to the nucleus to interact with DNA-bound proteins. This in turn activates the transcription of selected target genes such as Hes1{Upregulated by LSJL}, Hes5{Upregulated by LSJL}, and Hes7"
"Jagged1mediated Notch signaling in human bone marrow stem cells is necessary to initiate chondrogenesis but must be switched off for chondrogenesis to proceed"<-So ideally Notch signaling would be induced while performing LSJL and then downregulated when LSJL ceases.
According to Notch receptor and Notch ligand expression in developing avian cartilage., "abolition of Notch signalling is needed to allow full chondrogenesis to occur in human bone marrow stromal cells demonstrating that Notch signalling is not conserved between species."
Cartilage, SOX9 and Notch signals in chondrogenesis.
According to Notch receptor and Notch ligand expression in developing avian cartilage., "abolition of Notch signalling is needed to allow full chondrogenesis to occur in human bone marrow stromal cells demonstrating that Notch signalling is not conserved between species."
Cartilage, SOX9 and Notch signals in chondrogenesis.
"Cell rounding has been shown to help restore the chondrocyte phenotype"
"TFP medium contains transforming growth factor β1 (TGFβ1), fibroblast growth factor 2 (FGF-2) and platelet-derived growth factor BB (PDGF-BB), with 10% fetal bovine serum (FBS). In the presence of these three growth factors the chondrocytes divide more rapidly, they become spindle-shaped and demonstrate dramatically reduced contact inhibition. After culture in TFP the chondrocytes showed increased multipotency with some cells able to differentiate into osteoblasts and adipocytes as well as regaining a chondrocyte phenotype. Once transduced the cells expressed 10-fold higher levels of SOX9 and this level remained high as the cells were further passaged."<-so this study will show us whether elevated levels of Sox9 will help increase height regardless of how Sox9 degrades Beta-Catenin.
"the cells had an increased potential to respond to chondrogenic signals after they were transduced with SOX9"<-Maybe lack of Sox9 in epiphyseal bone marrow can lead to a reduced response to methods intended to induce epiphyseal chondrogenesis.
"In lowered oxygen conditions there is increased COL2A1 gene expression when chondrocytes are grown in 3D culture systems and that this involves increases in endogenous SOX9 expression"
In the study FGF2 increased expression of the Sox trio so FGF2 may be a potential way to induce Sox9 as a precursor to inducing chondrogenesis.
"Notch-2 receptor [is] broadly distributed throughout the epiphyseal growth plate"
According to Histone deacetylase inhibitors upregulate Notch-1 and inhibit growth in pheochromocytoma cells., valproic acid (VPA) and suberoyl bis-hydroxamic acid (SBHA) can activate Notch. So possibly use those chemicals to induce chondrogenesis and then wait for serum levels to decrease until Notch signaling is abolished to allow chondrogenesis to continue.
Beta-Catenin may help regulate Notch Signaling.
Beta-catenin modulates the level and transcriptional activity of Notch1/NICD through its direct interaction.
"Wnt and Notch1 signaling pathways play an important role in a variety of biological processes including embryonic induction, the polarity of cell division, cell fate, and cell growth. This report shows that beta-catenin can regulate the level and transcriptional activity of the Notch1 and Notch1 intracellular domain (NICD). The in vivo and in vitro results demonstrate that beta-catenin binds with Notch1 and NICD, for which its Armadillo repeat domain is essential. It was further demonstrated that beta-catenin could upregulate the level of Notch1 and NICD[interesting since Sox9 degrades beta-catenin which would decrease Notch1 and Notch1 is needed to induce chondrogenesis], possibly by competing the common ubiquitin-dependent degradation machinery. In addition, beta-catenin enhanced the transcriptional activity of NICD on the hairy and enhancer of split 1 (HES1) and CSL through its C-terminal transactivation domain. This effect of cooperative regulation by beta-catenin could also be observed in bone morphogenetic protein 2 (BMP2) induced osteogenic differentiation of C2C12 cells. beta-catenin coexpression with NICD enhanced the alkaline phosphatase (ALP) activity in C2C12 cells compared with either beta-catenin or NICD expression alone. Culturing C2C12 cells on Delta-1 coated dishes together with Wnt3-conditioned media induced noticeable increases in ALP staining, verifying that employed physiological levels of NICD and beta-catenin are sufficient to induce ALP activation. Furthermore, effects of beta-catenin on Notch1 were dramatically diminished by overexpressed LEF1[Maybe Sox9 degrades the Beta-Catenin earmarked for the TCF/LEF1 complex rather than NICD]. Overall, our data suggest that beta-catenin can act as a switching molecule between the classical TCF/LEF1 mediated pathway and NICD mediated pathway."
Beta-catenin modulates the level and transcriptional activity of Notch1/NICD through its direct interaction.
"Wnt and Notch1 signaling pathways play an important role in a variety of biological processes including embryonic induction, the polarity of cell division, cell fate, and cell growth. This report shows that beta-catenin can regulate the level and transcriptional activity of the Notch1 and Notch1 intracellular domain (NICD). The in vivo and in vitro results demonstrate that beta-catenin binds with Notch1 and NICD, for which its Armadillo repeat domain is essential. It was further demonstrated that beta-catenin could upregulate the level of Notch1 and NICD[interesting since Sox9 degrades beta-catenin which would decrease Notch1 and Notch1 is needed to induce chondrogenesis], possibly by competing the common ubiquitin-dependent degradation machinery. In addition, beta-catenin enhanced the transcriptional activity of NICD on the hairy and enhancer of split 1 (HES1) and CSL through its C-terminal transactivation domain. This effect of cooperative regulation by beta-catenin could also be observed in bone morphogenetic protein 2 (BMP2) induced osteogenic differentiation of C2C12 cells. beta-catenin coexpression with NICD enhanced the alkaline phosphatase (ALP) activity in C2C12 cells compared with either beta-catenin or NICD expression alone. Culturing C2C12 cells on Delta-1 coated dishes together with Wnt3-conditioned media induced noticeable increases in ALP staining, verifying that employed physiological levels of NICD and beta-catenin are sufficient to induce ALP activation. Furthermore, effects of beta-catenin on Notch1 were dramatically diminished by overexpressed LEF1[Maybe Sox9 degrades the Beta-Catenin earmarked for the TCF/LEF1 complex rather than NICD]. Overall, our data suggest that beta-catenin can act as a switching molecule between the classical TCF/LEF1 mediated pathway and NICD mediated pathway."
"In the presence of Wnt signaling, β-catenin is relieved from the destruction complex including APC/Axin/GSK3β, and the degradation pathway of β-catenin by the ubiquitination machinery is inhibited."
"In this condition, β-catenin accumulates in the cytoplasm and the nucleus where it interacts with the LEF1/TCF family transcription factors (LEF1, TCF-1, -3 and -4)"
"NICD has been reported to interact with LEF1, a transcriptional component of Wnt signaling, and has been suggested to function as a coactivator for LEF1. Presenilin and GSK3β are other proteins acting to meet Notch signaling and Wnt signaling at the cytoplasmic level. Presenilin has the activity of Notch cleavage into NICD, and is involved in the stability of β-catenin. Likewise, GSK-3β is involved in Wnt/β-catenin signaling and Notch signaling. β-catenin can be phosphorylated by GSK3β and regulated by the proteosome-dependent degradation machinery, and Notch2 is also associated with GSK3β, which inhibits the Notch2 activity"
"Notch1 and NICD directly bind to the arm repeat domain of β-catenin, resulting in reciprocal increases in the levels of Notch1 and β-catenin."
Repression of chondrogenesis through binding of notch signaling proteins HES-1 and HEY-1 to N-box domains in the COL2A1 enhancer site.
"Notch signaling is implicated in the repression of mesenchymal stem cell (MSC) chondrogenic differentiation.
Notch intracellular domain (NICD) protein levels were monitored via Western blotting throughout chondrogenic differentiation of human MSCs in pellet cultures. Overexpression of Notch signaling components and their effect on chondrogenesis was achieved by transfecting plasmids coding for NICD, HES-1, and HERP-2/HEY-1. COL2A1 and AGGRECAN expression was monitored via quantitative polymerase chain reaction analysis. Chromatin immunoprecipitation (ChIP) was used to test whether HES-1 and HEY-1 bind putative N-box domains in intron 1 of COL2A1.
High levels of NICD proteins were reduced during chondrogenesis of human MSCs, and this was mediated by transforming growth factor beta3 (TGFbeta3). COL2A1 gene expression was repressed following overexpression of NICD (2-fold) and HES-1 (3-fold) and was markedly repressed by overexpression of HEY-1 (80-fold). HEY-1 repressed AGGRECAN expression 10-fold, while NICD and HES-1 had no effect. We identified 2 putative N-box domains adjacent to, and part of, the SOX9 enhancer binding site located in intron 1 of COL2A1. ChIP studies showed that endogenous HES-1 and HEY-1 bound to these sites. Transducin-like enhancer, the HES-1 corepressor protein, was displaced during chondrogenic differentiation and following TGFbeta3 treatment.
Notch signaling proteins act on the SOX9 binding site in the COL2A1 enhancer and prevent SOX9-mediated transcriptional activation of COL2A1 and, thus, chondrogenic differentiation."
Repression of chondrogenesis through binding of notch signaling proteins HES-1 and HEY-1 to N-box domains in the COL2A1 enhancer site.
"Notch signaling is implicated in the repression of mesenchymal stem cell (MSC) chondrogenic differentiation.
Notch intracellular domain (NICD) protein levels were monitored via Western blotting throughout chondrogenic differentiation of human MSCs in pellet cultures. Overexpression of Notch signaling components and their effect on chondrogenesis was achieved by transfecting plasmids coding for NICD, HES-1, and HERP-2/HEY-1. COL2A1 and AGGRECAN expression was monitored via quantitative polymerase chain reaction analysis. Chromatin immunoprecipitation (ChIP) was used to test whether HES-1 and HEY-1 bind putative N-box domains in intron 1 of COL2A1.
High levels of NICD proteins were reduced during chondrogenesis of human MSCs, and this was mediated by transforming growth factor beta3 (TGFbeta3). COL2A1 gene expression was repressed following overexpression of NICD (2-fold) and HES-1 (3-fold) and was markedly repressed by overexpression of HEY-1 (80-fold). HEY-1 repressed AGGRECAN expression 10-fold, while NICD and HES-1 had no effect. We identified 2 putative N-box domains adjacent to, and part of, the SOX9 enhancer binding site located in intron 1 of COL2A1. ChIP studies showed that endogenous HES-1 and HEY-1 bound to these sites. Transducin-like enhancer, the HES-1 corepressor protein, was displaced during chondrogenic differentiation and following TGFbeta3 treatment.
Notch signaling proteins act on the SOX9 binding site in the COL2A1 enhancer and prevent SOX9-mediated transcriptional activation of COL2A1 and, thus, chondrogenic differentiation."
"Hes-1{upregulated in LSJL} and Hey-1 bind N-box domains in the enhancer region of Col2a1. Since this site is required for Sox9 mediated transcription of Col2a1, we propose that bHLH proteins negatively compete with Sox9 binding to the enhancer site and prevent Sox9-mediated transcriptional activation of Col2a1 and thus chondrogenic differentiation."<-So it's likely a bad thing that Hes-1 increased in expression however Sox9 increased in expression 0.5 fold higher than Hes-1.
Notch gain of function inhibits chondrocyte differentiation via Rbpj-dependent suppression of Sox9.
"Sustained Notch activation in cartilage leads to chondrodysplasia[short stature]. Genetic evidence indicates that Notch regulates limb bud mesenchymal stem cell differentiation into chondrocytes via an Rbpj-dependent Notch pathway. Cartilage specific Notch gain-of-function (GOF) mutant mice display chondrodysplasia accompanied by loss of Sox9 expression in vertebrae. To evaluate the contribution of an Rbpj-dependent Notch signaling to this phenotype, we deleted Rbpj on the Notch GOF background. These mice showed persistent spine abnormalities characterized by "butterfly" vertebrae suggesting that removal of Rbpj does not fully rescue the axial skeleton deformities caused by Notch GOF. However, Sox9 protein level was restored in Rbpj deficient Notch GOF mice compared to Notch GOF mutants, demonstrating that regulation of Sox9 expression is canonical or Rbpj-dependent. The association of the Rbpj/NICD complex with the Sox9 promoter is associated with transcriptional repression of Sox9 in a cellular model of chondrocyte differentiation. Notch negatively regulates chondrocyte differentiation in the axial skeleton by suppressing Sox9 transcription, and Rbpj-independent Notch signaling mechanisms may also contribute to axial skeletogenesis."
"The signaling from Notch ligands to Notch receptors is initiated by the release of the active Notch intracellular domain (NICD) through proteolytic events mediated by Presenilin1/2 within the gamma-secretase complex."
"NICD forms a complex with Rbpj to activate the transcription of downstream targets including the Hes and Hey family of genes"
"loss of function (LOF) in DLL3 or JAGGED1, both Notch ligands, or loss of function in HES7, LFNG or MESP2 [can result in short stature]"
"Over-expression of the Notch ligand DLL1 in chick limb buds blocked differentiation from pre-hypertrophic to hypertrophic chondrocytes and resulted in shortened skeletal elements with decreased expression of Col2a1 and Col10a1."
"increased expression of Ihh and Col10a1 with an elongated hypertrophic zone [occurred] when Presenilin1/ 2 [was] deleted in limb bud mesenchymal progenitor cells"
"an elongated hypertrophic zone resulted from deletion of Notch1/2 receptors by Prx1-cre"
"In the earlier progenitor pool, Notch signaling promotes chondrocyte proliferation. In committed chondrocytes Notch inhibits chondrocyte differentiation and maturation."
"canonical Notch targets Hey1 and Hey2{upregulated with LSJL} were up-regulated in the GOF mice"
"Notch receptor physically interacts with activated Beta-catenin to constrain the accumulation of Beta-catenin in stem or progenitor cells"
Notch Suppresses Nuclear Factor of Activated T Cells (Nfat) Transactivation and Nfatc1 Expression in Chondrocytes.
"Notch1 to Notch4 transmembrane receptors determine cell fate, and release of the Notch intracellular domain (NICD) in the cytoplasm induces gene expression. Notch regulates endochondral ossification, . Nuclear factor of activated T cells (Nfatc) transcription factors regulate chondrogenesis, and we asked whether Notch modifies Nfat signaling in chondrocytes. Notch was induced in teratocarcinoma ATDC5 chondrogenic cells infected with a retroviral vector, where the cytomegalovirus (CMV) promoter directs NICD expression. NICD suppressed chondrocyte differentiation and inhibited Nfat transactivation and Nfatc1 expression. Notch was activated in chondrocytes from Rosa(Notch) mice, where the Rosa26 promoter is upstream of a loxP-flanked STOP cassette and NICD. To excise the STOP cassette and express NICD, Rosa(Notch) chondrocytes were infected with an adenoviral vector where the CMV promoter directs Cre expression (Ad-CMV-Cre). Notch1 and Notch2 mediate the effects of Notch in skeletal cells, and to inhibit Notch signaling, chondrocytes from mice homozygous for Notch1 and Notch2 alleles targeted with loxP sites were infected with Ad-CMV-Cre. NICD suppressed chondrogenic nodules formation and expression of selected chondrocyte gene markers, induced Col10a1{up} and Mmp13, and suppressed Nfat transactivation and Nfatc1 expression, whereas inactivation of Notch1 and Notch2 did not affect chondrocyte differentiation. To investigate Nfatc1 function in chondrocytes, Nfatc1 was induced in Rosa(Notch) chondrocytes overexpressing NICD or controls. Nfatc1 suppressed chondrocyte differentiation and opposed Col10a1 induction by Notch. Notch suppresses Nfat transactivation in chondrocytes and Notch and Nfatc1 regulate chondrocyte differentiation."
"In the Notch canonical signaling pathway,NICD translocates to the nucleus and interacts with Epstein-Barr virus latencyC promoter binding factor 1, Suppressor of hairless and Lag-1 (Csl), also known as Rbpj in mice, a DNA-binding protein that suppresses gene expression by recruiting repressors of transcription."
"Overexpression of NICD in vitro suppresses the differentiation of chondrogenic cells, and Notch signaling inhibition in limb bud cell cultures enhances chondrogenesis"
"NICD overexpression in mesenchymal cells suppresses chondrogenesis"
"inactivation of Csl in chondrocytes causes elongation of the hypertrophic zone"
"The phosphatase calcineurin induces Nfat transactivation by dephosphorylating specific serine residues in the SRR and SPXXrepeat motifs of the regulatory domain of Nfat"
"Dephosphorylated Nfat translocates to the nucleus and induces expression of Nfat target genes, such as the isoform of Regulator of calcineurin 1 transcribed from the promoter region upstream of exon 4 (Rcan1.4). Phosphorylation of the SRR and SPXX repeat motifs prevents association to DNA and induces Nfat nuclear export, resulting in inhibition of Nfat transactivation. Nfatc1 to Nfatc4 are expressed in murine chondrocytes, and Nfatc2-null mice display ectopic chondrogenesis and joint abnormalities due to hypertrophic differentiation of articular chondrocytes"
"a constitutively active Nfatc3{down} mutant promoted chondrogenic differentiation of mesenchymal cells in vitro"
"activation of Nfatc2 during early chondrogenesis of limb bud cell cultures leads to enhanced chondrocyte differentiation, whereas Nfatc2 activation in more mature cells prevented further differentiation"
In ATDC5 cells: "NICD suppressed Col10a1, Mmp13, and Vegf transcripts" but it did not inhibit early markers like Sox9 and Col2a1 significantly but it did affect early markers Sox9 and Ihh non-significantly but in a noticeable way. NICD also upregulated Hey2{up} and Hes1{up}.
Notch signaling in chondrocytes modulates endochondral ossification and osteoarthritis development.
"Intracellular domains of Notch1 and -2 were translocated into the nucleus of chondrocytes with their differentiation in mouse limb cartilage and in mouse and human OA articular cartilage. A tissue-specific inactivation of the Notch transcriptional effector recombination signal binding protein for Ig kappa J (RBPjκ) in chondroprogenitor cells of SRY-box containing gene 9 (Sox9)-Cre;Rbpj(fl/fl) mouse embryos caused an impaired terminal stage of endochondral ossification in the limb cartilage. The RBPjκ inactivation in adult articular cartilage after normal skeletal growth using type II collagen (Col2a1)-Cre(ERT);Rbpj(fl/fl) mice by tamoxifen injection caused resistance to OA development in the knee joint. Notch intracellular domain with the effector RBPjκ stimulated endochondral ossification through induction of the target gene Hes1{up} in chondrocytes. Among the Notch ligands, Jagged1 was strongly induced during OA development. Finally, intraarticular injection of N-[N-(3,5-diflurophenylacetate)-l-alanyl]-(S)-phenylglycine t-butyl ester (DAPT), a small compound Notch inhibitor, to the mouse knee joint prevented OA development. The RBPjκ-dependent Notch signaling in chondrocytes modulates the terminal stage of endochondral ossification."
"Although the conditional knockout (Sox9-Cre;Rbpjfl/fl) mice died shortly after birth, they showed a slight dwarfism during the embryonic periods compared with the Rbpjfl/fl littermates: the limbs and vertebrae were about 5–10% shorter in Sox9-Cre;Rbpjfl/fl mice than in these littermates"
"the percentage of the hypertrophic zone relative to the limb length was much increased, whereas that of the bone area was considerably decreased in the Sox9-Cre;Rbpjfl/fl limbs, indicating that the RBPjκ knockout impaired the terminal differentiation stage in such aspects as matrix degradation and vascular invasion"
"Mmp13 and Vegfa, as well as Hes1, were decreased by the RBPjκ knockout"
Notch gain of function inhibits chondrocyte differentiation via Rbpj-dependent suppression of Sox9.
"Sustained Notch activation in cartilage leads to chondrodysplasia[short stature]. Genetic evidence indicates that Notch regulates limb bud mesenchymal stem cell differentiation into chondrocytes via an Rbpj-dependent Notch pathway. Cartilage specific Notch gain-of-function (GOF) mutant mice display chondrodysplasia accompanied by loss of Sox9 expression in vertebrae. To evaluate the contribution of an Rbpj-dependent Notch signaling to this phenotype, we deleted Rbpj on the Notch GOF background. These mice showed persistent spine abnormalities characterized by "butterfly" vertebrae suggesting that removal of Rbpj does not fully rescue the axial skeleton deformities caused by Notch GOF. However, Sox9 protein level was restored in Rbpj deficient Notch GOF mice compared to Notch GOF mutants, demonstrating that regulation of Sox9 expression is canonical or Rbpj-dependent. The association of the Rbpj/NICD complex with the Sox9 promoter is associated with transcriptional repression of Sox9 in a cellular model of chondrocyte differentiation. Notch negatively regulates chondrocyte differentiation in the axial skeleton by suppressing Sox9 transcription, and Rbpj-independent Notch signaling mechanisms may also contribute to axial skeletogenesis."
"The signaling from Notch ligands to Notch receptors is initiated by the release of the active Notch intracellular domain (NICD) through proteolytic events mediated by Presenilin1/2 within the gamma-secretase complex."
"NICD forms a complex with Rbpj to activate the transcription of downstream targets including the Hes and Hey family of genes"
"loss of function (LOF) in DLL3 or JAGGED1, both Notch ligands, or loss of function in HES7, LFNG or MESP2 [can result in short stature]"
"Over-expression of the Notch ligand DLL1 in chick limb buds blocked differentiation from pre-hypertrophic to hypertrophic chondrocytes and resulted in shortened skeletal elements with decreased expression of Col2a1 and Col10a1."
"increased expression of Ihh and Col10a1 with an elongated hypertrophic zone [occurred] when Presenilin1/ 2 [was] deleted in limb bud mesenchymal progenitor cells"
"an elongated hypertrophic zone resulted from deletion of Notch1/2 receptors by Prx1-cre"
"In the earlier progenitor pool, Notch signaling promotes chondrocyte proliferation. In committed chondrocytes Notch inhibits chondrocyte differentiation and maturation."
"canonical Notch targets Hey1 and Hey2{upregulated with LSJL} were up-regulated in the GOF mice"
"Notch receptor physically interacts with activated Beta-catenin to constrain the accumulation of Beta-catenin in stem or progenitor cells"
Notch Suppresses Nuclear Factor of Activated T Cells (Nfat) Transactivation and Nfatc1 Expression in Chondrocytes.
"Notch1 to Notch4 transmembrane receptors determine cell fate, and release of the Notch intracellular domain (NICD) in the cytoplasm induces gene expression. Notch regulates endochondral ossification, . Nuclear factor of activated T cells (Nfatc) transcription factors regulate chondrogenesis, and we asked whether Notch modifies Nfat signaling in chondrocytes. Notch was induced in teratocarcinoma ATDC5 chondrogenic cells infected with a retroviral vector, where the cytomegalovirus (CMV) promoter directs NICD expression. NICD suppressed chondrocyte differentiation and inhibited Nfat transactivation and Nfatc1 expression. Notch was activated in chondrocytes from Rosa(Notch) mice, where the Rosa26 promoter is upstream of a loxP-flanked STOP cassette and NICD. To excise the STOP cassette and express NICD, Rosa(Notch) chondrocytes were infected with an adenoviral vector where the CMV promoter directs Cre expression (Ad-CMV-Cre). Notch1 and Notch2 mediate the effects of Notch in skeletal cells, and to inhibit Notch signaling, chondrocytes from mice homozygous for Notch1 and Notch2 alleles targeted with loxP sites were infected with Ad-CMV-Cre. NICD suppressed chondrogenic nodules formation and expression of selected chondrocyte gene markers, induced Col10a1{up} and Mmp13, and suppressed Nfat transactivation and Nfatc1 expression, whereas inactivation of Notch1 and Notch2 did not affect chondrocyte differentiation. To investigate Nfatc1 function in chondrocytes, Nfatc1 was induced in Rosa(Notch) chondrocytes overexpressing NICD or controls. Nfatc1 suppressed chondrocyte differentiation and opposed Col10a1 induction by Notch. Notch suppresses Nfat transactivation in chondrocytes and Notch and Nfatc1 regulate chondrocyte differentiation."
"In the Notch canonical signaling pathway,NICD translocates to the nucleus and interacts with Epstein-Barr virus latencyC promoter binding factor 1, Suppressor of hairless and Lag-1 (Csl), also known as Rbpj in mice, a DNA-binding protein that suppresses gene expression by recruiting repressors of transcription."
"Overexpression of NICD in vitro suppresses the differentiation of chondrogenic cells, and Notch signaling inhibition in limb bud cell cultures enhances chondrogenesis"
"NICD overexpression in mesenchymal cells suppresses chondrogenesis"
"inactivation of Csl in chondrocytes causes elongation of the hypertrophic zone"
"The phosphatase calcineurin induces Nfat transactivation by dephosphorylating specific serine residues in the SRR and SPXXrepeat motifs of the regulatory domain of Nfat"
"Dephosphorylated Nfat translocates to the nucleus and induces expression of Nfat target genes, such as the isoform of Regulator of calcineurin 1 transcribed from the promoter region upstream of exon 4 (Rcan1.4). Phosphorylation of the SRR and SPXX repeat motifs prevents association to DNA and induces Nfat nuclear export, resulting in inhibition of Nfat transactivation. Nfatc1 to Nfatc4 are expressed in murine chondrocytes, and Nfatc2-null mice display ectopic chondrogenesis and joint abnormalities due to hypertrophic differentiation of articular chondrocytes"
"a constitutively active Nfatc3{down} mutant promoted chondrogenic differentiation of mesenchymal cells in vitro"
"activation of Nfatc2 during early chondrogenesis of limb bud cell cultures leads to enhanced chondrocyte differentiation, whereas Nfatc2 activation in more mature cells prevented further differentiation"
In ATDC5 cells: "NICD suppressed Col10a1, Mmp13, and Vegf transcripts" but it did not inhibit early markers like Sox9 and Col2a1 significantly but it did affect early markers Sox9 and Ihh non-significantly but in a noticeable way. NICD also upregulated Hey2{up} and Hes1{up}.
Notch signaling in chondrocytes modulates endochondral ossification and osteoarthritis development.
"Intracellular domains of Notch1 and -2 were translocated into the nucleus of chondrocytes with their differentiation in mouse limb cartilage and in mouse and human OA articular cartilage. A tissue-specific inactivation of the Notch transcriptional effector recombination signal binding protein for Ig kappa J (RBPjκ) in chondroprogenitor cells of SRY-box containing gene 9 (Sox9)-Cre;Rbpj(fl/fl) mouse embryos caused an impaired terminal stage of endochondral ossification in the limb cartilage. The RBPjκ inactivation in adult articular cartilage after normal skeletal growth using type II collagen (Col2a1)-Cre(ERT);Rbpj(fl/fl) mice by tamoxifen injection caused resistance to OA development in the knee joint. Notch intracellular domain with the effector RBPjκ stimulated endochondral ossification through induction of the target gene Hes1{up} in chondrocytes. Among the Notch ligands, Jagged1 was strongly induced during OA development. Finally, intraarticular injection of N-[N-(3,5-diflurophenylacetate)-l-alanyl]-(S)-phenylglycine t-butyl ester (DAPT), a small compound Notch inhibitor, to the mouse knee joint prevented OA development. The RBPjκ-dependent Notch signaling in chondrocytes modulates the terminal stage of endochondral ossification."
"Although the conditional knockout (Sox9-Cre;Rbpjfl/fl) mice died shortly after birth, they showed a slight dwarfism during the embryonic periods compared with the Rbpjfl/fl littermates: the limbs and vertebrae were about 5–10% shorter in Sox9-Cre;Rbpjfl/fl mice than in these littermates"
"the percentage of the hypertrophic zone relative to the limb length was much increased, whereas that of the bone area was considerably decreased in the Sox9-Cre;Rbpjfl/fl limbs, indicating that the RBPjκ knockout impaired the terminal differentiation stage in such aspects as matrix degradation and vascular invasion"
"Mmp13 and Vegfa, as well as Hes1, were decreased by the RBPjκ knockout"
"Under the surgical OA induction, however, the cartilage degradation as well as expressions of Mmp13, Vegfa, and Hes1 were suppressed in the Col2a1-CreERT;Rbpjfl/fl knee joints, compared with the Rbpjfl/fl joints, confirming the resistance to OA development by the RBPjκ insufficiency in adult articular cartilage."<-interesting this indicates that osteoarthritis may be due to endochondral ossification of articular cartilage than due to degeneration of chondrocytes into fibroblasts.
"In the cultures of articular chondrocytes, treatment with DAPT inhibited Mmp13, Vegfa, and Hes1 expressions and also alkaline phosphatase and Alizarin red stainings"
No comments:
Post a Comment