Monday, August 23, 2010

Wnt pathway in chondrogenesis

WNT elements are involved very highly in LSJL.  Wnt2, frillzed 2, 3, Wnt-1 inducible protein 2.  Wnt's and Dkk's not enumerated.

Comprehensive expression analysis of all Wnt genes and their major secreted antagonists during mouse limb development and cartilage differentiation.

"Members of the family of secreted frizzled related proteins (Sfrp) directly bind to Wnt ligands and thus preventing receptor binding"

Wnt2b has been detected (weakly) in postnatal cartilage growth plates and in mesenchymal tissues.

"Wnt5b was expressed in perichondrium, prehypertrophic and hypertrophic chondrocytes"<-Wnt5a has similar expression patterns.

"Wnt11 was expressed in the mesenchyme of developing limb buds"<-LSJL altered the frizzled receptor expression of Wnt11.  Wnt11 was also expressed in prehypertrophic chondrocytes.

Sfrp 1,3, 5 were expressed in both the mesenchyme and chondrocytes(these are Wnt antagonists)  It's unclear whether they were expressed during LSJL.

The nuclear hormone receptor PPARγ counteracts vascular calcification by inhibiting Wnt5a signalling in vascular smooth muscle cells.

"Deletion of the nuclear receptor PPARγ in vascular smooth muscle cells of low density lipoprotein receptor (LDLr)-deficient mice fed an atherogenic diet high in cholesterol, accelerates vascular calcification with chondrogenic metaplasia within the lesions. Vascular calcification in the absence of PPARγ requires expression of the transmembrane receptor LDLr-related protein-1 in vascular smooth muscle cells. LDLr-related protein-1 promotes a previously unknown Wnt5a-dependent prochondrogenic pathway. PPARγ protects against vascular calcification by inducing the expression of secreted frizzled-related protein-2, which functions as a Wnt5a antagonist."

Wnt gene expression in the post-natal growth plate: regulation with chondrocyte differentiation.

"Six [Wnt's] were expressed in postnatal growth plate. Of these, Wnts -2b, -4, and -10b signal through the canonical beta-catenin pathway and Wnts -5a, -5b, and -11 signal through the noncanonical calcium pathway. The spatial expression for these six Wnts was remarkably similar, showing low mRNA expression in the resting zone, increasing expression as the chondrocytes differentiated into the proliferative and prehypertrophic state and then (except Wnt-2b) decreasing expression as the chondrocytes underwent hypertrophic differentiation. Wnt signaling modulates chondrocyte proliferation and hypertrophic differentiation. mRNA expression of these Wnt genes persisted at similar levels at 4 weeks, when longitudinal bone growth is waning."

"The resting zone, adjacent to the epiphyseal bone, contains small, roughly spherical chondrocytes distributed singly or in pairs{so maybe condensation is not required to form new growth plates}, which divide infrequently and are considered "stem-like cells" capable of generating new clones of proliferative chondrocytes"

"Wnt-10a and Wnt-7b which were detected at very low levels in growth plate but not detected in bone or perichondrium"

"Genetic inactivation of β-catenin stimulates ectopic formation of chondrocytes"

"Wnt-4 overexpression accelerates hypertrophy of chondrocytes. However, mice null for Wnt-4 have been reported to have either no growth plate phenotype"

"Wnt-5a null mice display a severe skeletal phenotype with limb truncation."

"Overexpression of Wnt-11 in the developing chick limb results in slightly truncated limbs and joint fusion but does not appear to delay chondrocyte differentiation"

"Dermal fibroblasts cultured in the presence of chondroinductive demineralized bone powder show increased expression of Wnt-2b, Wnt-5b, and Wnt-10b "

"Wnts that had been readily detected in the growth plates of 1-week-old mice were still expressed in growth plate cartilage of 4-week-old mice, at similar levels."<-Wnts may not be involved in growth plate senescence.

WNT5A regulates chondrocyte differentiation through differential use of the CaN/NFAT and IKK/NF-kappaB pathways.

"Wnt5a treatment of chondroprogenitor cells increased chondrocyte hypertrophy and was associated with an increase in nuclear factor of activated T cells (NFAT) and a decrease in nuclear factor-kappaB (NF-kappaB) activation. Wnt5a inhibited chondrocyte hypertrophy. This inhibition of hypertrophy occurred with the reciprocal signaling activation, in that a decrease in NFAT and an increase in NF-kappaB activation was observed. The increase in chondroprogenitor cell differentiation with Wnt5a treatment was blocked by calmodulin kinase or NFAT loss of function. The repression of chondrocyte hypertrophy observed was abrogated by NF-kappaB loss of function. Activation of the NFAT pathway downstream of Wnt5a negatively regulated NF-kappaB activity. Wnt5a acts to increase chondrocyte differentiation at an early stage through calmodulin kinase /NFAT-dependent induction of Sox9. Wnt5a represses chondrocyte hypertrophy via NF-kappaB-dependent inhibition of Runx2 expression."<So Wnt5a likely can help with LSJL but not with developed growth plates.

"In noncanonical Wnt calcium-dependent signaling, activation of phospholipase C through a G protein-dependent mechanism facilitates a rise in intracellular calcium concentrations. This increase in calcium then leads to the activation of several known calcium-sensitive effectors, including calmodulin kinase (CaMK), calcineurin (CaN), cAMP response element-binding protein (CREB), and protein kinase C"

"Wnt5a has been shown to specifically promote entry into the prehypertrophic phase, whereas it conversely blocks chondrocyte hypertrophy"

"nuclear factor of activated T cells (NFAT)4 [induces] chondrogenesis"<-LSJL downregulates NFAT4 as NFATC3.

The effects of Wnt inhibitors on the chondrogenesis of human mesenchymal stem cells.

"The inhibition of the Wnt pathway promotes chondrogenesis of human mesenchymal stem cells (hMSCs). In vitro pellet cultures were prepared using MSCs at passage 3 at varying concentrations of 100, 200, and 300 ng/mL of either Dickkopf (DKK)-1 or secreted frizzled-related protein (sFRP)-1, and analyzed for chondrogenic gene and protein expressions after 3 and 6 days of culture. To study the effects on chondrogenesis at a longer term, 200 ng of sFRP-1 was challenged either in the presence or absence of transforming growth factor (TGF)-beta(3) to pellets of MSCs at passage 3 for 7 days. Pellets were cultured without sFRP-1 for 14 further days. For early chondrogenesis, both DKK-1 and sFRP-1 increased glycosaminoglycan synthesis as well as the gene and protein expressions of SOX-9 and type II collagen, more prominently by sFRP-1 than by DKK-1{LSJL upregulates Dkk3}. After 21 days of in vitro chondrogenic culture under TGF-beta(3), sFRP-1 treatment did not further increase the gene expression of SOX-9 and type II collagen{this is likely the point where hypertrophic differentiation began to dominate and other transcription factors took over for Sox9}."

"Ectopic canonical Wnt signaling led to enhanced ossification and suppression of chondrocyte formation during the developmental process. Conversely, genetic inactivation of β-catenin caused ectopic formation of chondrocytes at the expense of osteoblast differentiation during both intramembranous and endochondral ossification."

"By conditionally deleting β-catenin in the limb and head mesenchyme, the osteoblast precursors lacking β-catenin were blocked from undergoing differentiation, and instead developed into chondrocytes. Their loss- and gain-of-function analyses also revealed that canonical Wnt signaling was essential for skeletal lineage differentiation, preventing transdifferentiation of osteoblastic cells into chondrocytes."

"Dickkopf (DKK)-1, inhibits Wnt signaling by binding to LRP5/6 and Kremens, a single pass transmembrane protein, forming a tertiary complex that is subsequently internalized and degraded."

"DKK-1 and sFRP-1 did not significantly affect the β-catenin gene expression. On the other hand, findings from western blotting showed that the β-catenin protein expression significantly decreased with the treatment of either DKK-1 (10%) or sFRP-1"

"When the TGF-β3 was introduced for the chondrogenic culture for longer term, sFRP-1 did not change the chondrogenic gene expression profiles. TGF-β3 overwhelmed the effect of β-catenin, making the additional treatment of Wnt inhibitors redundant."<-so it may be better to upregulate TGF-Beta than use Wnt inhibitors due to Wnt pathway's importance in hypertrophic stage of endochondral ossification.

WNT Signaling and Cartilage: Of Mice and Men.

"Chondrocytes that sparsely reside in the matrix and rarely proliferate are the key cellular mediators for cartilage homeostasis. WNT signaling pathway [is involved in] articular cartilage [maintenance]. Various WNTs are involved in the subsequent stages of chondrocyte differentiation during development, and deregulation of WNT signaling was observed in cartilage degeneration. Even though gene expression and protein synthesis can be activated upon injury, articular cartilage has a limited ability of self-repair and efforts to regenerate articular cartilage have so far not been successful. Because WNT signaling was found to be involved in the development and maintenance of cartilage as well as in the degeneration of cartilage, interfering with this pathway might contribute to improving cartilage regeneration."

Wnt pathway regulation by demineralized bone is approximated by both BMP-2 and TGF-β1 signaling.

"Allogeneic demineralized bone is used extensively as a clinical graft material because it has osteo/chondroinductive and osteoconductive properties. Demineralized bone powder (DBP) induces chondrogenic differentiation of human dermal fibroblasts (hDFs) in three-dimensional collagen cultures, but the initiating mechanisms have not been fully characterized nor has it been shown that bone morphogenetic proteins (BMPs) recapitulate DBP's effects on target cells. Among the many signaling pathways regulated in hDFs by DBP prior to in vitro chondrogenesis, there are changes in Wnts and their receptors that may contribute to DBP actions. This study tests the hypothesis that DBP modulation of Wnt signaling entails both BMP and TGF-β pathways. We compared the effects of DBP, TGF-β1, or BMP-2 on Wnt signaling components in hDFs by Wnt signaling macroarray, RT-PCR, in situ hybridization, and Western immunoblot analyses. Many effects of DBP on Wnt signaling components were not shared by BMP-2, and likewise DBP effects on Wnt genes and β-catenin only partially required the TGF-β pathway, as shown by selective inhibition of TGF-β/activin receptor-like kinase. The analyses revealed that 64% (16/25) of the Wnt signaling components regulated by DBP were regulated similarly by the sum of effects by BMP-2 and by TGF-β1. In conclusion, signaling mechanisms of inductive DBP in human dermal fibroblasts involve the modulation of multiple Wnt signals through both BMP and TGF-β pathways."

"All three agents upregulated Wnt2{up}, Wnt5a, Fzd2{up}, Fzd7, and Gja1, and all three downregulated Msx2"

PTGS2-Upregulated in LSJL and by TGF-Beta.  Downregulated by DBP and BMP-2.  Col1a1 up in LSJL and upregulated by all three.  Egr1 down in all three but up in LSJL.

Reduced expression of Sfrp1 during chondrogenesis and in articular chondrocytes correlates with osteoarthritis in STR/ort mice.

"To circumvent the problems of genetic and environmental diversity hampering the analysis in humans, we turned to a murine model for human knee osteoarthritis (OA) and fine mapped a previously defined OA-quantitative trait locus (QTL). We here focused on one of the candidate genes within the OA-QTL encoding the Wnt antagonist secreted frizzled related protein 1 (Sfrp1). Sequence analysis of the Sfrp1 gene in the OA strain STR/ort revealed 23 polymorphic changes with a potential to alter the gene expression. A reduced expression in STR/ort mice was demonstrated for articular chondrocytes and hypertrophic chondrocytes of the femoral growth plate. In vitro generated mesenchymal stem cells (MSC) and chondrogenically differentiated MSC (cMSC) [had] reduced Sfrp1 expression in STR/ort mice. This reduced Sfrp1 expression in MSC correlated with an increased amount of cytoplasmic β-catenin, a downregulation of the Wnt target gene Pparγ and an upregulation of Runx2 as well as a preferential differentiation of the MSC along the osteoblasts lineage {So possibly Sfrp1 is pro-chondrogenic}. Given the role of Wnt signalling during chondrogenesis and in maintaining the integrity of the long lived articular chondrocytes, we conclude that the reduced Sfrp1 expression in STR/ort mice not only leads to an increased activation of the Wnt/ß-catenin signalling early in life but also renders the articular cartilage prone to premature ageing and to the development of OA."

"Activation and overexpression of β-catenin in adult articular chondrocytes induced hypertrophy and cell death resembling OA"

"Fgfr1{up}was shown to be expressed during MSC expansion and during chondrocyte hypertrophy in enchondral ossification and [may] be important for cell survival and extra cellular matrix production"

Wnt-mediated reciprocal regulation between cartilage and bone development during endochondral ossification.

"Given their independent function, a requirement for β-catenin is not the same as that for Wnt. We investigated the effect of Wnt proteins in both tissues through generating cartilage- or bone-specific Wls null mice, respectively. Depletion of Wls by Col2-Cre, which would block Wnt secretion in the chondrocytes and perichondrium, delayed chondrocyte hypertrophy in the growth plate and impaired perichondrial osteogenesis. Loss of Wls in chondrocytes also disturbed the proliferating chondrocyte morphology and division orientation, which was similar to the defect observed in Wnt5a null mice. On the other hand, inactivation of Wls in osteoblasts by Col1-Cre resulted in a shorter hypertrophic zone and an increase of TRAP positive cell number in the chondro-osseous junction of growth plate, coupled with a decrease in bone mass. Wnt proteins not only modulate differentiation and cellular communication within populations of chondrocytes, but also mediate the cross regulation between the chondrocytes and osteoblasts in growth plate."

"CanonicalWnt/Beta-catenin signaling determines the chondrogenic or osteogenic commitment from mesenchymal progenitor cells at early stage, and promotes the hypertrophic chondrocyte differentiation at late stage"

"non-canonical Wnt5a and Wnt5b differentially regulate the transition of chondrocyte differentiation within the growth plate. The gradient of Wnt5a/PCP signaling instructs the directional limb outgrowth by establishing polarity in chondrocytes"

"Wntless (Wls), or Gpr177, is a trafficking protein that is essential for the secretion of both canonical and non-canonical Wnt proteins"

"canonical Wnt signaling is extensively active in growth plate chondrocytes"

Conditional expression of Wnt4 during chondrogenesis leads to dwarfism in mice.

"A mouse Wnt4 cDNA was introduced into the ubiquitously expressed Rosa26 (R26) locus by gene targeting in embryonic stem (ES) cells. The expression of Wnt4 from the R26 locus was blocked by a neomycin selection cassette flanked by loxP sites (floxneo) that was positioned between the Rosa26 promoter and the Wnt4 cDNA, creating the allele designated R26(floxneoWnt4). Wnt4 expression was activated during chondrogenesis using Col2a1-Cre transgenic mice that express Cre recombinase in differentiating chondrocytes. R26(floxneoWnt4); Col2a1-Cre double heterozygous mice exhibited a growth deficiency, beginning approximately 7 to 10 days after birth, that resulted in dwarfism. In addition, they also had craniofacial abnormalities, and delayed ossification of the lumbar vertebrae and pelvic bones. Histological analysis revealed a disruption in the organization of the growth plates and a delay in the onset of the primary and secondary ossification centers. Wnt4 overexpression caused decreased proliferation and altered maturation of chondrocytes. In addition, R26(floxneoWnt4); Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF).  Wnt4 overexpression leads to dwarfism in mice.  Decreased VEGF expression suggests that defects in vascularization may contribute to the dwarf phenotype."

"Wnt4 accelerates chondrocyte differentiation, whereas Wnt5a inhibits this process"

"Misexpression of Wnt5b as well as Wnt5a inhibits chondrogenesis in mice, but they appear to act differently. Wnt5a inhibits the transition from resting to proliferating chondrocytes in the growth plate, whereas Wnt5b promotes this transition as well as chondrocyte proliferation"

"At 9 months of age, the tibiae of mutants were deficient in bone marrow and were filled with adipocytes in epiphyseal and metaphyseal regions. In contrast, inspection of 12-month-old control mice showed metaphyseal regions full of bone marrow"

FGF, TGFβ and Wnt crosstalk: embryonic to in vitro cartilage development from mesenchymal stem cells.

"members of the fibroblast growth factor (FGF), transforming growth factor-β (TGFβ) and wingless-type (Wnt) protein families are involved in controlling different differentiation stages during chondrogenesis."

"at multiple stages Smad7 inhibits chondrocyte differentiation"

"Inhibition of the p38–MAPK pathway resembles the effect of Wnt-7a misexpression in that N-cadherin expression is sustained and chondrogenesis inhibited."

"Chondrocyte maturation is reliant on an increase in Runx2 levels, accompanied by a decrease in Twist1 levels"

"disruption of Axin2 signalling-accelerated chondrocyte maturation"
"TGFβ signalling has been shown to promote cellular senescence [and stimulates proliferation as well] of MSCs through G1 arrest, achieved through activation of cyclin D kinase inhibitors (p16, p21 and p53). FGF-2, through inhibition of TGFβ2 expression, was able to suppress this cellular senescence"
"FGF-2 reduces the multipotentiality of MSCs, instead priming them for the chondrogenic lineage. "

Priming 3D cultures of human mesenchymal stromal cells towards cartilage formation via developmental pathways.

"Wnt3a and FGF2 induce hBM-MSC to proliferate in 3D culture as an undifferentiated pool of progenitors (defined by clonogenic capacity and expression of typical markers), retaining chondrogenic potential upon induction by suitable morphogens. hBM-MSC were responsive to Wnt signaling in 3D pellet culture, as assessed by significant upregulation of main target genes and increase of unphosphorylated β catenin levels. Wnt3a was able to induce a 5-fold increase in the number of proliferating hBM-MSC (6.4% vs 1.3% in the vehicle condition), although total DNA content of the 3D construct was decreasing over time. Preconditioning with Wnt3a improved TGF-β1 mediated chondrogenesis (30% more glycosaminoglycans/cell in average). In contrast to developmental and 2D MSC culture models, FGF2 antagonized the Wnt-mediated effects. Interestingly, the CD146+ subpopulation was found to be more responsive to Wnt3a."

"Upon chondrogenic and hypertrophic activation, the endochondral process proceeds in a semi-autonomous and self-regulated manner, comparable to normal embryonic development and leading to efficient hBM-MSC induction towards the osteoblastic lineage"

"Wnt exposure maintains mesenchymal cells in a proliferative state, while the exit from the signal, along the elongation of the tissue (i.e. increase in distance from Wnt source), allows for the chondrogenic differentiation to occur"

Genes activated by the Wnt pathway in the cell lines were Axin2, Dkk1, Cyclin D1, and p21.  The Wnt group also had a significantly greater portion of phosphorylated Beta-Catenin.  The Wnt treated group had more cells that underwent cell division.

Following TGF-Beta induction of chondrogenesis, the Wnt pretreated group was more chondrogenic than the non-treated group.  Chondroinduction did not occur without TGF-Beta induction.  FGF2 antagonized the beneficial affects of Wnt3a.

"the expansion of mesenchymal progenitors densely packed in 3D nodules, which precedes the complete chondrogenic differentiation of human embryonic stem cells, requires the stimulation by Wnt3a and FGF2, along with Activin-A and/or BMP4."

"hBM-MSC markedly differ from limb mesenchymal cells, so that their expansion and chondrogenesis cannot be instructed by directly recapitulating developmental cues"

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