LSJL Pathway Analysis

This was obtained using Partek Pathway software with the excel spreadsheet with list of 2 fold changers.  mm8 of whole mouse genome was selected.  Targeted pathways with relevance to longitudinal bone growth or chondroinduction were selected.  The old pathways generated in the previous gene expression study are included for reference.  The closest thing to a chondroinduction pathway is TGF-Beta.  These pathways can be used to predict phosphorylation and additional gene expression changes.  Green means expression was altered.  Light green means more downregulated whereas darker more redish green means more significantly upregulated.  Most important to note is that LSJL upregulates several genes in cell adhesion which is an important precursor to chondrogenesis(3.3% of genes).  LSJL also upregulates genes involved in cartilage condensation, chondrobast differentiation, and chondrocyte differentiation which are three processes that would be key to formation of new growth plates with p < 0.05 and containing at least 20% of the genes involved in that process expressed at over 2-fold levels.

Comparing this to axial loading genes(Alternative Splicing in Bone Following Mechanical Loading), many processes are shared except that the processes of chondrocyte development, cartilage development in endochondral bone morphogenesis, skeletal system morphogenesis, growth plate cartilage development, and endochondral ossification are significantly altered by LSJL whereas they are not by axial loading(see below for definitions).  It is likely that these 5 processes are part of the reason that LSJL can increase height whereas axial loading does not.  

I don't believe cartilage development in endochondral bone morphogenesis is a key different as the only gene that's present in LSJL and not in axial loading is Col1a1 and that is not a master regulator gene.  But the absence of Col1a1 stimulation in axial loading is odd.  Growth Plate Cartilage Development is a process where axial loading is missing Col9a1 and THBS3.  THBS3 is a gene that could potentially have a big impact.

In Skeletal System Morphogenesis most of the genes are shared except HHIP, Col11a1(Col11a2 is altered by axial loading however), Tgfbr1, and Wwox are altered by LSJL but not by axial loading.  HHIP is a key gene so that is likely the differentiating factor.

In endochondral ossification, Col10a1, Col1a1, and GNAS are three genes altered by LSJL and not by axial loading.

So GNAS, HHIP, and THBS3 may be key genes explaining part of the reason why LSJL can increase height but axial loading does not.

Surprisingly, axial loading significantly stimulates key processes in initial growth plate formation like chondroblast differentiation, cartilage condensation, and chondrocyte differentiation.  Chondrocyte development was very significantly enriched by axial loading but the key gene Sox9 was missing. Therefore, axial loading must fail at a later stage in the process likely at inducing Sox9.  Since the cutoff was much lower for the axial loading study than the LSJL study it's possible that LSJL alters many of the genes by the axial loading study as well.

Axial loading may very well induce the early stages of chondrogenesis and there may in fact be ectopic cartilage(or chondrocyte) formation in response to mechanical loading.  However, that cartilage seems to fail to become a growth plate.  To test this hypothesis we'd need to see if ectopic chondrocytes do form under the bone under Beta-Catenin knockout ectopic chondrocytes occur under the perichondrium(immature periosteum).  If we can find evidence that axial loading can induce ectopic chondrocyte formation in adult bone that would be a step forward in proving LSJL.

Pathways(not all are below) that LSJL upregulates with p < 0.05:
ECM-Receptor
Protein Digestion
Focal Adhesion
Amoebiasis
Mineral Absorption
Hedgehog Signaling
Aminoacyl-tRNA
Caffeine Metabolism
Olfactory Transduction
Homologous Recombination

Gene Ontology(calculated using Partek) for Chondroinduction Related Gene Sets:

The Enrichment Score is Next to the data.  The higher the enrichment score the better.  Multiples of genes are intentional.  Bolded means that p < 0.05.  % is the precent of genes in gene group that are present.

Positive Regulation of Cartilage Development(4.33):<-"Any process that increases the rate, frequency, or extent of cartilage development, the process whose specific outcome is the progression of the cartilage over time, from its formation to the mature structure."
Cyr61
Smad1{down}
Sox9
Sox9

Chondrocyte Differentiation(3.98):<-"The process in which a chondroblast acquires specialized structural and/or functional features of a chondrocyte." 20%
Col2a1
Fgfr1
Fgfr1
Mef2d{down}
Osr1{down}

Cartilage Condensation(8.77):<-"The condensation of mesenchymal cells that have been committed to differentiate into chondrocytes." 35.29%
Acan
Barx2
Barx2
BMPR1B
Col11a1
Col2a1
Sox9
Sox9

Full Gene List.

Chondroblast Differentiation(4.17):<-"The process in which a mesenchymal cell, acquires specialized structural and/or functional features of a chondroblast." 50%
Cyr61
FGF2

Full Gene List.  RARA downregulation is key, all others need to be upregulated.  FGF-4 downregulation may also be important.

Cell Adhesion Molecule Binding(9.04):<-"Interacting selectively and non-covalently with a cell adhesion molecule."
Adam8{down}
Anp32a{down}
Cadm1{down}
CD226{down}
CD226{down}
FGFR1
FGFR1
PVRL3
Sdcbp
Vcam1{down}

Positive regulation of Chondrocyte Differentiation(1.69):<-"Any process that activates or increases the frequency, rate or extent of chondrocyte differentiation. "
Gli3
Gli3
Sox9
Sox9

Regulation of Cell Shape(2.30):<-Chondrocytes are round shaped.  "Any process that modulates the surface configuration of a cell."
Epb4.1
Fgd4{down}
Gna13{down}
IL6
KDR
PDPN
PDPN

Bone morphogenesis(1.50):<-"The process in which bones are generated and organized. "
Cdx1{down}
Pax1

Endochondral Bone Morphogenesis(no enrichment score unfortunately):<-"The process in which bones are generated and organized as a result of the conversion of initial cartilaginous anlage into bone. "

Skeletal system morphogenesis(4.88):<-"The process in which the anatomical structures of the skeleton are generated and organized."
Col11a1
Col11a1
Col2a1
HHIP
MMP2
TGFBR1{down}
WWOX{down}

Cartilage Development(6.72):<-"The process whose specific outcome is the progression of the cartilage over time, from its formation to the mature structure. Cartilage is a connective tissue dominated by extracellular matrix containing collagen type II and large amounts of proteoglycan, particularly chondroitin sulfate."
BMPR1B
COL10A1
COL11A1
COL2A1
COL9A1
EDN1
GNAS{down}
PRRX1
PRRX1
PRRX2
SOX9
SOX9

Cartilage development involved in Endochondral bone morphogenesis(3.03):<-"The process whose specific outcome is the progression of the cartilage that will provide a scaffold for mineralization of endochondral bones. "
Col1a1
Col2a1

Growth Plate Cartilage Development(3.03):<-"The process whose specific outcome is the progression of the cartilage that will provide a scaffold for mineralization of endochondral bones as they elongate or grow. "
Col9a1
THBS3

Endochondral Ossification(6.54):<-"Replacement ossification wherein bone tissue replaces cartilage. "
Col10a1
Col1a1
GNAS
GNAS{Down}
MEF2D{down}

Stem Cell Differentiation(0.39):<-"The process in which a relatively unspecialized cell acquires specialized features of a stem cell. A stem cell is a cell that retains the ability to divide and proliferate throughout life to provide progenitor cells that can differentiate into specialized cells. "
Osr1{down}

Cell Adhesion Molecule Binding(9.04):<-"Interacting selectively and non-covalently with a cell adhesion molecule. "
Adam8{down}
Anp32a{down}
Cadm1{down}
CD226{down}
CD226{down}
Fgfr1
Fgfr1
Pvrl3
Sdcbp
Vcam1{down}

Actin Cytoskeleton Organization(4.43):<-"A process that is carried out at the cellular level which results in the assembly, arrangement of constituent parts, or disassembly of cytoskeletal structures comprising actin filaments and their associated proteins."  Reorganization of the actin cytoskeleton is essential for chondrogenic differentiation
Diap3{down}
Enah{down}
Epb4.1
Fhod1{down}
Fhod3
Fscn1
Kras{down}
Pfn2
Shc1{down}
Tesk2{down}
Wasf2{down}

Skeletal System Development(6.87):<-"The process whose specific outcome is the progression of the skeleton over time, from its formation to the mature structure. The skeleton is the bony framework of the body in vertebrates (endoskeleton) or the hard outer envelope of insects (exoskeleton or dermoskeleton)."
BMPR1B
COL1A1
COL2A1
COL5A2
EDN1
GNAS
GNAS{DOWN}
HEXA{DOWN}
HOXD10
PAX1
SOX9
SOX9
TGFBR1{DOWN}
WDR5{DOWN}

Positive Regulation of Cell Migration(9.41): Stem Cell Migration is required to form new growth plates.  "Any process that activates or increases the frequency, rate or extent of cell migration."
Adam8{DOWN}
C3AR1
ccl24
Cdh13
Col18a1
Col18a1
Col1a1
Cyr61
EDN1
EGFR
GRB7{DOWN}
HBEGF
IRS1{DOWN}
KDR
MAPK8{DOWN}
PDPN
PDPN
TRIP6{DOWN}

Chondrocyte Development(3.80):<-"The process whose specific outcome is the progression of a chondrocyte over time, from its commitment to its mature state. Chondrocyte development does not include the steps involved in committing a chondroblast to a chondrocyte fate."
Acan
Col11a1
Sox9
Sox9

Homophilic Cell Adhesion(8.52)<-Required for prechondrogenic mesenchymal condensation.
Chondroblast differentiation is the strongest proof of chondroinduction by LSJL.  "The attachment of an adhesion molecule in one cell to an identical molecule in an adjacent cell. "
Cadm1{down}
Cadm3
Cdh10
Cdh11{down}
Cdh13
Cdh15{down}
Chl1
Dsc2
Dsg4{down}
Frem2
Pvrl3
Robo2

Tissue Remodeling(8.36)<-To make room for new growth plates..."The reorganization or renovation of existing tissues. This process can either change the characteristics of a tissue such as in blood vessel remodeling, or result in the dynamic equilibrium of a tissue such as in bone remodeling." 57.14%
CSPG4
CSPG4
LIPA{down}
MMP14
MMP2

Other possibly related pathways(genes not enumerated with p < 0.01, possible involvement with endochondral ossification, and enrichment score >1):

Reponse to Zinc Ion("Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a zinc ion stimulus. ")

Inflammatory Response("The immediate defensive reaction (by vertebrate tissue) to infection or injury caused by chemical or physical agents. The process is characterized by local vasodilation, extravasation of plasma into intercellular spaces and accumulation of white blood cells and macrophages. ")

Ras Protein Signal Transduction("A series of molecular signals within the cell that are mediated by a member of the Ras superfamily of proteins switching to a GTP-bound active state. ")

PI3K Cascade("A series of reactions, mediated by the intracellular phosphatidylinositol 3-kinase (PI3K). PI3K cascades lie downstream of many cell surface receptor linked signaling pathways and regulate numerous cellular functions. ")

Wnt Receptor Signaling Pathway("The series of molecular signals initiated by binding of a Wnt protein to a frizzled family receptor on the surface of the target cell and ending with a change in cell state. ")

Centriole Replication("The cell cycle process in which a daughter centriole is formed perpendicular to an existing centriole. An immature centriole contains a ninefold radially symmetric array of single microtubules; mature centrioles consist of a radial array of nine microtubule triplets, doublets, or singlets depending upon the species and cell type. ")

transcription from RNA polymerase II promoter("The synthesis of RNA from a DNA template by RNA polymerase II, originating at an RNA polymerase II promoter. Includes transcription of messenger RNA (mRNA) and certain small nuclear RNAs (snRNAs). ")

Protein autophosphorylation("The phosphorylation by a protein of one or more of its own amino acid residues, or residues on an identical protein. ")

Endocytosis("A vesicle-mediated transport process in which cells take up external materials or membrane constituents by the invagination of a small region of the plasma membrane to form a new membrane-bounded vesicle. ")

Apoptosis("A programmed cell death process which begins when a cell receives an internal (e.g. DNA damage) or external signal (e.g. an extracellular death ligand), and proceeds through a series of biochemical events (signaling pathways) which typically lead to rounding-up of the cell, retraction of pseudopodes, reduction of cellular volume (pyknosis), chromatin condensation, nuclear fragmentation (karyorrhexis), plasma membrane blebbing and fragmentation of the cell into apoptotic bodies. The process ends when the cell has died. The process is divided into a signaling pathway phase and into an execution phase, which is triggered by the former. ")

Multicellular organism growth("The increase in size or mass of an entire multicellular organism, as opposed to cell growth. ")

Positive regulation of endothelial cell proliferation("Any process that activates or increases the rate or extent of endothelial cell proliferation. ")

Positive regulation of mesenchymal cell proliferation

negative regulation of transcription from RNA polymerase II promoter

positive regulation of cell proliferation

negative regulation of cell proliferation

negative regulation of cell adhesion

negative regulation of apoptosis

Positive regulation of inflammatory response("Any process that activates or increases the frequency, rate or extent of the inflammatory response. ")


positive regulation of transcription from RNA polymerase II promoter

positive regulation of angiogenesis

positive regulation of cell differentiation

peptide hormone processing(endocrine hormones)("The generation of a mature peptide hormone by posttranslational processing of a prohormone.")

Collagen fibril organization("The generation of a mature peptide hormone by posttranslational processing of a prohormone.")

Alpha-tubulin binding("Interacting selectively and non-covalently with the microtubule constituent protein alpha-tubulin. ")

Beta-tubulin binding

Integrin binding

protein C-terminis binding("Interacting selectively and non-covalently with a protein C-terminus, the end of any peptide chain at which the 1-carboxy function of a constituent amino acid is not attached in peptide linkage to another amino-acid residue. ")

histone acetylase binding

histone deacetylase binding

fibronectin binding

misfolded protein binding

epidermal growth factor receptor binding

neurexin family protein binding

platelet-derived growth factor binding

PDZ domain binding

Heparin Binding

Hyaluronic Acid binding

Calcium ion binding

Magnesium ion binding

UDP-N-acetylmuramate dehydrogenase activity("Catalysis of the reaction: UDP-N-acetylmuramate + NADP+ = UDP-N-acetyl-3-O-(1-carboxyvinyl)-D-glucosamine + NADPH + H+. ") NADPH is required for chondrogenesis.

Endocytic vesicle("A membrane-bounded intracellular vesicle formed by invagination of the plasma membrane around an extracellular substance. Endocytic vesicles fuse with early endosomes to deliver the cargo for further sorting. ")

Spliceosomal complex("Any of a series of ribonucleoprotein complexes that contain RNA and small nuclear ribonucleoproteins (snRNPs), and are formed sequentially during the splicing of a messenger RNA primary transcript to excise an intron. ")

Cellular response to calcium ion

Nitric Oxide Mediated Signal Transduction

Peptidyl-tyrosine dephosphorylation("The removal of phosphoric residues from peptidyl-O-phospho-tyrosine to form peptidyl-tyrosine.")

Gene Expression analysis from Life Map Discovery:(Genes that are uporegulated in LSJL and also that cell lineage.  Bold are selective markers.  - means that the cell type is characterized by a lack of that protein.

The apical ectodermal ridge (AER) is a structure that forms from the ectodermal cells at the distal end of each limb bud and acts as a major signaling center to ensure proper development of a limb.


Zeugopod growth plate is used.  The zeugopod growth plates are located at both ends of the long bones (ulna, radius, tibia and fibula):

Aldh1a3{down}
Angptl1
Bmp2
Car12
Col10a1
Epyc
Fgf2
Fgfr1
Kctd4
MMP14
Smad1{down}
Smpd3{down}
Sox9
Bsp
Zcchc5

Progress Zone cells: Progress zone cells are rapidly proliferating, undifferentiated mesenchyme cells, situated directly underneath the Apical Ectodermal Ridge.

Hoxd10
Bmp2
Fgfr1

Prechondrocytic mesenchymal cells:  Prechondrocytic mesenchymal cells are mesenchymal cells ready to acquire osteochondral fate. They produce an extracellular matrix rich in hyaluronan.

Col1a1
-Sox9
Lin28b
Pkia
Slitrk6
Tax1bp1{down}

Mesenchymal condensate cells:  Mesenchymal condensate cells are mesenchymal cells that aggregate at future sites of bone formation at the initiation of chondrogenesis. The aggregation step is essential for further osteochondral differentiation.

Acan
Col11a1
Col2a1
Sox9
Bmpr1b
Bmp2
Fgf2
Fgfr1
Gli3
Thbs2
Aspn
Brpf3{down}
Ccng2{down}
Cd9{down}
Cops2{down}
Ebf3{down}
Egfr
Flnc
Gas1
Hapln1
Htra1
Jun
Lum
Matn3
Moxd1
Myl1
Osr1{down}
Sstr4{down}
Sulf1
Thbs4
Vcan
Zcchc5
1110012J17Rik

Zeugopod prechondrocytes: Prechondrocytes are resting, non-proliferative cells.

Acan
Col11a1
Col9a1
Sox9
Fgf2
Thbs2

Zeugopod epiphyseal end chondrocytes: Chondrocytes are highly proliferative columnar cells.

Acan
Col2a1
Sox9
Matn3
Hapln1
Bmp2
Gli3
Lepre1
Tgfbr1 {down}
Arsi
Cgref1
D10Bwg1379e
Dock8 {down}
Epha3
Epyc
Fry {down}
Matn2
Plekha6 {down}
Smpd3{down}
Tcf7 {down}
Thbs4

Prehypertrophic Chondrocytes: Prehypertrophic chondrocytes are chondrocytes at maturation, which no longer proliferate.
Col10a1
Fgfr1

Hypertrophic Chondrocytes: Hypertrophic chondrocytes are non-proliferative cells, larger in size than prehypertrophic chondrocytes and synthesize a characteristic cartilogenous matrix.

Col10a1
Bsp
Sox9
Bmp2
Fgf2
Fgfr1
Smad1 {down}

Terminal Chondrocytes: Terminal chondrocytes undergo apoptosis and are replaced by bone cells. They have an osteoblast-like phenotype.

MMP14
Bsp

Focal Adhesion and Chondroinduction

Focal Adhesion is a statistically significant pathway involved in LSJL.  Whether LSJL stimulates or disrupts focal adhesion is unclear.

Cytoskeletal and Focal Adhesion Influences on Mesenchymal Stem Cell Shape, Mechanical Properties, and Differentiation Down Osteogenic, Adipogenic, and Chondrogenic Pathways.

"Osteogenic differentiation is more prevalent in MSCs with a stiff, spread actin cytoskeleton and greater numbers of focal adhesions. Both adipogenic differentiation and chondrogenic differentiation are encouraged when MSCs have a spherical morphology associated with a dispersed actin cytoskeleton with few focal adhesions."

"uniaxial[all along one axis like all along the lateral axis like with LSJL], unconfined compression and cyclic hydrostatic pressure increase chondrogenesis."

"If the perinuclear actin cap is inhibited, the formation of actin stress fibers in hMSC is prevented."

"chondrocytes have a stiffness that is midway between MSCs and adipocytes at 1.2 kPa."

"the actin cytoskeleton is not as important for mechanotransduction in chondrocytes as it is in osteoblasts."

" In chick wing-bud MSCs, disrupting the actin cytoskeleton with cytochalasin-D encouraged chondrogenesis. Various factors known to regulate the actin cytoskeleton play an important role in regulating chondrogenesis. As MSCs undergo chondrogenesis, they exhibit a decrease in RhoA activity. This decrease in RhoA is at least partially responsible for chondrogenic differentiation of MSCs, as MSCs made to overexpress RhoA exhibited decreased chondrogenesis. This shows that the cell causes the actin cytoskeleton to become more diffused through decreased RhoA activity to undergo chondrogenesis."

"Cytoskeletal configuration and regulation have also been shown to play an important role in chondrogenesis. Like adipogenesis, chondrogenesis is encouraged by having a rounded cell shape. MSCs plated on surfaces modified with RGD (arginine, glycine, aspartic acid) peptides spread out, whereas MSCs on RGE (arginine, glycine, glutamic acid) peptide-modified surfaces, which prevent focal adhesion attachment, remain rounded. MSCs on the RGD surfaces showed decreased chondrogenesis as evidenced by lower levels of mRNA for collagen II and aggrecan. These surfaces also exemplified the importance of interactions between integrins and the actin cytoskeleton in chondrogenesis. While cells seeded on RGD surfaces had high levels of localized vinculin expression, MSCs on RGE surfaces expressed only low levels of vinculin that were not localized. This implies that strong focal adhesion attachments are not necessary or beneficial to chondrogenesis."

"Active mechanical interventions can also cause actin–cytoskeletal alignment in MSCs. In 3D culture, MSCs show actin–cytoskeletal alignment parallel to the compression direction both in cyclic and static unconfined compression. Because the pellets are unconfined, there is likely an element of tensile strain in the direction perpendicular to applied compression. Therefore, it is not surprising that cyclic uniaxial stretch can also be used to cause actin cytoskeletal alignment in MSCs. We have shown that actin fiber alignment in the direction of applied strain occurs in hMSC exposed to uniaxial cyclic tensile strain."

Microgravity reduces RhoA organization and actin organization.  Chondrogenesis is encouraged by reduced spreading and a disrupted actin cytoskeleton.  DC electric fields also encourage a chondrogenic round cell shape.

"Oscillatory fluid flow, which has been shown to increase cytoskeletal organization, has also been reported to increase Sox9 production in MSCs when delivered at 1 Hz with a peak force of 1 Pa."<-so the correlation between cytoskeleton organization and chondroinduction is not perfect.

Less focal adhesions are more conductive to chondrogenesis.

MicroRNA-488 suppresses cell migration through modulation of the focal adhesion activity during chondrogenic differentiation of chick limb mesenchymal cells.

"we investigated the role of miRNA-488 in the cellular condensation, which is essential initiation for chondrogenic differentiation. We found that miRNA-488 expression is up-regulated at the precondensation stage and then down-regulated at the postcondensation stage. Blockade of miRNA-488 via the use of PNA (peanut agglutinin)-based ASOs (antisense oligonucleotides) decreased the protein level of integrins β1 and phosphorylated FAK (focal adhesion kinase) and resulted in the suppression of cell motility and migration. Moreover, in parallel with theses observation, treatment of anti-miRNA-488 oligonucleotides up-regulated the level of MMP (matrix metalloprotease)-2 activity, and co-treatment with GM6001, an MMP inhibitor, induced recovery of cellular condensation inhibited by blockade of miRNA-488. Collectively, our results suggest that miRNA-488 is one of regulator in cell to ECM (extracellular matrix) interaction through modulation of focal adhesion activity by MMP-2 during chondrogenesis of limb mesenchymal cells."

Curcumin inhibits cellular condensation and alters microfilament organization during chondrogenic differentiation of limb bud mesenchymal cells.

"Curcumin is a well known natural polyphenol product isolated from the rhizome of the plant Curcuma longa, anti-inflammatory agent for arthritis by inhibiting synthesis of inflammatory prostaglandins. Treating chick limb bud mesenchymal cells with curcumin suppressed chondrogenesis by stimulating apoptotic cell death. It also inhibited reorganization of the actin cytoskeleton into a cortical pattern concomitant with rounding of chondrogenic competent cells and down-regulation of integrin beta1 and focal adhesion kinase (FAK) phosphorylation. Curcumin suppressed the phosphorylation of Akt leading to Akt inactivation. Activation of Akt by introducing a myristoylated, constitutively active form of Akt reversed the inhibitory actions of curcumin during chondrogenesis. In summary, for the first time, we describe biological properties of curcumin during chondrogenic differentiation of chick limb bud mesenchymal cells. Curcumin suppressed chondrogenesis by stimulating apoptotic cell death and down-regulating integrin-mediated reorganization of actin cytoskeleton via modulation of Akt signaling."

"The anti-proliferative effects of curcumin include blocks at specific cell cycle stages, particularly a G2/M block"

"Curcumin is known to inhibit a number of kinase and enzyme activities, including PKC, phosphorylase kinase, EGF receptor{up in LSJL} and erbB2"

"curcumin suppressed chondrogenesis by stimulating apoptotic cell death, down-regulating integrin β1{LSJL upregulates ITGB-Like 1, structurally it is similar to ITGB1 but functionally it is unclear} and lowering the phosphorylation level of FAK. This, in turn, lead to reorganization of the actin cytoskeleton via modulation of Akt signaling."

"members of the integrin family, including the α5β1 fibronectin receptor, α2β1 and α3β1, and the vitronectin receptor αvβ3 interact reversibly with both ECM proteins and cytoskeletal structures"

"Akt signaling positively regulates chondrogenic differentiation of chick limb bud mesenchymal cells via reorganization of the actin cytoskeleton to a cortical pattern with concomitant rounding of chondrogenic competent cells"<-LSJL increases Akt-phosphorylation.

"phosphorylation level of Akt was decreased when cells were exposed to curcumin"

" Undifferentiated chondrogenic progenitor cells are characterized by pronounced fibrillar actin fibers, whereas chondrocytes display a primarily cortical organization of their actin filaments "

Focal adhesion kinase/Src suppresses early chondrogenesis: central role of CCN2.

"FAK/Src signaling, which mediates cell/matrix attachment, suppresses early chondrogenesis, including the induction of Ccn2, Agc{up in LSJL}, and Sox6. The FAK/Src inhibitor PP2 elevates Ccn2, Agc, and Sox6 expression in wild-type mesenchymal cells in micromass culture, but not in cells lacking CCN2."

"The level and efficiency of overall chondrogenic differentiation, and hence subsequent bone development, is directly correlated to the density of the initial condensation"

" the formation of condensations and cartilage nodules requires adhesive signaling and remodeling of the actin cytoskeleton, via Rho and Rac"

WISP2, Cyr61, and Acan are upregulated in CCN2+/+ versus CCN2-/- as well as in LSJL.

"In the Fak-/- cells, L-Sox5 and Col2a1 mRNA expression was significantly higher than in the wild-type controls"

"CCN2 is a key modulator of adhesive signaling and promotes chondrogenic gene expression in vivo and in vitro"

"FAK signaling may suppress chondrogenesis by preventing the induction of CCN2."


Identification and characterization of the integrin alpha2beta1 binding motif in chondroadherin mediating cell attachment.

"Chondroadherin is a leucine-rich repeat protein known to mediate adhesion of isolated cells via the integrin α(2)β(1) and to interact with collagen. In this work, we show that cell adhesion to chondroadherin leads to activation of MAPKs but does not result in cell spreading and division. This is in contrast to the spreading and dividing of cells grown on collagen, although the binding is mediated via the same α(2)β(1) receptor. We identified a cell binding motif, CQLRGLRRWLEAK(318) by mass spectrometry after protease digestion of chondroadherin. Cells adhering to the synthetic peptide CQLRGLRRWLEAK(318) remained round, as was observed when they bound to the intact protein. The peptide added in solution was able to inhibit cell adhesion to the intact protein in a dose-dependent manner and was also verified to bind to the α(2)β(1) integrin. A cyclic peptide, CQLRGLRRWLEAKASRPDATC(326), mimicking the structural constraints of this sequence in the intact protein, showed similar efficiency in inhibiting binding to chondroadherin. The unique peptide motif responsible for cellular binding is primarily located in the octamer sequence LRRWLEAK(318). Binding of cells to the active peptide or to chondroadherin immobilized on cell culture plates rapidly induces intracellular signaling (i.e. ERK phosphorylation). Thus, chondroadherin interaction with cells may be central for maintaining the adult chondrocyte phenotype and cartilage homeostasis. The peptides, particularly the more stable cyclic peptide, open new opportunities to modulate cell behavior in situations of tissue pathology."

"Aggrecan binds specifically to hyaluronan via its N-terminal domain, thereby forming multimolecular aggregates consisting of 100 or more molecules. This creates a very high fixed charge density, allowing for water retention and resistance to deformation"

"Cells adhering via α2β1 to chondroadherin remain round, as do cells adhering via α5β1 to the integrin binding domain of fibronectin. In the case of fibronectin, treatment with a heparin-binding fragment of the molecule (HepII), a peptide from this domain, or phorbol esters to activate protein kinase C is necessary to provide sufficient signals inducing spreading, formation of focal adhesions, and stress fibers. This process involves both integrin and syndecan receptors at the cell surface. Similarly, cell spreading can be induced on chondroadherin as a consequence of phorbol ester treatment"

"cell surface collagens mediate adhesion to matrilin-1. Binding of cells to chondroadherin was partially decreased upon collagenase treatment"

"anti-β1 integrin antibody prevented spreading, and the cells formed aggregates"

"the addition of EDTA, known to dissociate integrin interactions, lead to cell detachment"

"collagen type II (adhering to the α1β1, α2β1, α10β1, and α11β1 integrins), fibronectin (adhering primarily to the α5β1 integrin), laminin (adhering to the α6β1 integrin), vitronectin (adhering to the αVβ3 integrin), and chondroadherin (interacting only with the α2β1 integrin)."

"Adhesion of human chondrocytes to chondroadherin or peptide induces phosphorylation of ERK1/2."

MMP-2 functions as a negative regulator of chondrogenic cell condensation via down-regulation of the FAK-integrin beta1 interaction.

"we investigated the functional role of MMP-2 in chondrogenesis of leg bud mesenchymal cells. Small interference RNA (siRNA)-mediated knockdown of mmp-2{LSJL upregulates MMP2, maybe an MMP2 inhibitor would augment LSJL results} promoted precartilage condensation and chondrogenesis. Treatment with bafilomycin A1, an MMP-2 activator, or GM6001, an MMP inhibitor, at the pre-condensation stage resulted in the inhibition or promotion of chondrogenesis, respectively. By comparison, treatment at the post-condensation stage had little or no effect on chondrogenesis. These results indicate that MMP-2 is involved in the regulation of cell condensation. Inhibition of MMP-2 activity by mmp-2 specific siRNA increased the protein level of fibronectin, and integrins alpha5 and beta1. The interaction between focal adhesion kinase (FAK) and integrin beta1 leading to tyrosine phosphorylation of FAK was also enhanced. Moreover, inactivation of p38MAPK down-regulated the level of MMP-2 mRNA and activity, and increased mesenchymal cell condensation in parallel with enhanced phosphorylation of FAK. Taken together, our data indicate that MMP-2 mediates the inhibitory signals of p38MAPK during mesenchymal cell condensation by functioning as a negative regulator of focal adhesion activity regulated by FAK via interactions with fibronectin through integrin beta1."

"testican-1, an inhibitor of pro-MMP-2 activation, is expressed in joint and growth plate cartilage"

"the activity of MT1–MMP on the cell surface is constitutively down-regulated through a vacuolar H+-ATPase-dependent degradation process."

"Blockade of this degradation caused the accumulation of TIMP-free active MT1–MMP molecules on the cell surface and pro-MMP-2 activation was strongly enhanced as a consequence of this impaired degradation."

"FAK is localized to focal adhesions where it is activated by phosphorylation after integrin-mediated cell attachment"

"The tyrosine phosphorylation of FAK increased after knockdown of mmp-2, but decreased after activation of MMP-2"

"Adhesion of chondrocytes to the site of interaction between fibronectin and integrin β1 leads to tyrosine phosphorylation of cytoskeletal and signaling proteins localized at focal adhesions, including paxillin, c-Src and FAK"

"MMP-2 inhibits cellular condensation by down-regulating the integrin β1-mediated interaction between fibronectin and FAK, and tyrosine phosphorylation of focal adhesion components, such as FAK, paxillin and c-Src at the focal adhesion site. The p38MAPK pathway induces the down-regulation of tyrosine phosphorylation of FAK, via the up-regulation of MMP-2, at the transcription and protein activity levels, leading to inhibition of leg bud mesenchymal cell condensation."