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