Monday, September 28, 2009


Odz3 is upregulated by LSJL.

Expression of Ten-m/Odz3 in the fibrous layer of mandibular condylar cartilage during postnatal growth in mice.

"Mandibular condyle develops via the differentiation of the fibroblast-like cells covering the condyle into chondrocytes. We used laser-capture microdissection and cDNA microarray analysis to elucidate the genes that are highly expressed in these fibroblast-like cells. Among these genes, the transcription of Ten-m/Odz3 was significantly increased in the fibroblast-like cells compared with other cartilage tissues. For the first time, we describe the temporal and spatial expression of Ten-m/Odz3 mRNA in relation to the expression of type I, II, and X collagen mRNA, as determined by in-situ hybridization in mouse mandibular condylar cartilage and mouse femoral cartilage during the early stages of development. Ten-m/Odz3 was expressed in the fibrous layer and the proliferating and mature chondrocyte layers, which expressed type I and II collagen, respectively, but was not detected in the hypertrophic chondrocyte layer. Furthermore, we evaluated the in-vitro expression of Ten-m/Odz3 using ATDC5 cells, a mouse chondrogenic cell line. Ten-m/Odz3 was expressed during the early stage of the differentiation of mesenchymal cells into chondrocytes. These findings suggest that Ten-m/Odz3 is involved in the differentiation of chondrocytes and that it acts as a regulatory factor in the early stages of the development of mandibular condylar cartilage."

"Ten-m/Odz3 is expressed in prechondrogenic mesenchymal cells and may be directly or indirectly involved in the early stage of chondrogenic differentiation."

"inflammatory and/or mechanical stress may also modulate Ten-m/Odz3 expression in synobial fibroblasts in the temporomandibular joint."

Sunday, September 27, 2009

Noriaki Ono: Potential Height Increase ally

Here's a grant submitted by Noriaki Ono:


"The goal of this research project is to reveal stem/progenitor cells in the chondrocyte and osteoblast lineage in terms of their origin, distribution, regulated kinetics and genetic profiles in vivo.
Specific Aim 1. Stem-like chondrocytes at the top of the postnatal epiphyseal growth plate cartilage: In endochondral bone formation, chondrocytes in the specific regions termed growth plates continue to proliferate postnatally, providing engines for bone lengthening. Slowly dividing cells at the top [of] the growth plate probably share some characteristics of postnatal stem cells. First, existence of self-renewing chondrocytes that are the sources of all other chondrocytes in the growth plate will be demonstrated by a lineage-tracking experiment using a chondrocyte-specific inducible CreERt and a fluorescent reporter system with a long chase period. Second, the genetic make-up of label-retaining cells at the top of the growth plate will be characterized based on cDNA microarrays{If they're significantly different from stem cells then that's bad for adult height growth, the more similar they are the more promising evidence it provides for adult height growth.  Although adult stem cells have already been shown to have potential for chondrogenic differentiation.}. A chondrocyte-specific pulse-chase experiment will be performed to identify slowly replicating cells based on a doxycycline-regulatable Tet-off system and a histone 2B-bound EGFP (H2B-EGFP) label. Label-retaining and non-label-retaining chondrocytes will be isolated by fluorescent activated cell sorting (FACS). Genes specifically expressed in label-retaining chondrocytes will be tested for their gene expression during development by in situ hybridization, using probes identified in microarray experiments comparing the label-retaining and rapidly proliferating chondrocytes{This study will be extremely helpful}.
Specific Aim 3. Common stem/progenitor cells of the chondrocyte and the osteoblast lineage and their function: Inducible CreERt BAC transgenic mouse in which CreERt expression is regulated by the promoter of one of the commonly upregulated genes of Aim 1 and 2 will be created. To understand the role of these cells during skeletal development, the CreERt mice will be crossed with inducible diphtheria toxin receptor (iDTR) mice. Diphtheria toxin will be administered at various times of development, and disruption on normal skeletogenesis will be monitored to elucidate the role of these progenitors in vivo.."

Monday, September 21, 2009


ePHa5 is upregulated 2.65 fold by LSJL.

MicroRNA-34a regulates migration of chondroblast and IL-1β-induced degeneration of chondrocytes by targeting EphA5.

"expression of miR-34a increased upon chondrogenic inhibition. Blockade of miR-34a via PNA-based antisense oligonucleotides (ASOs) recovered the chondro-inhibitory actions of JNK inhibitor on migration of chondrogenic progenitors and the formation of precartilage condensation. Furthermore, we determined that EphA5 is a relevant target of miR-34a during chondrogenesis. MiR-34a was necessary and sufficient to down-regulate EphA5 expression, and up-modulation of EphA5 is sufficient to overcome inhibitory actions of miR-34 inhibition on cell migration and condensation of chick limb mesenchymal cells on collagen substrate."

EphA5 which is upregulated with LSJL is associated with chondrogenic migration and condensation.

"the protein level of type II collagen, a typical marker for chondrogenic differentiation, decreased when cells were exposed to JNK inhibitors"<-the upregulation of ePHa5 is also indicative that LSJL induces the JNK pathway.

"Down-regulation of cell migration by JNK inhibitor was overcome by co-treatment of miR-34a inhibitor under inactivation condition of endogenous EphA receptors."

"MiR-34 is known to target Notch, HMGA2, and Bcl-2"

Saturday, September 19, 2009

B2A peptide

B2A peptide induces chondrogenic differentiation in vitro and enhances cartilage repair in rats.

"This study investigated whether the synthetic peptide B2A (B2A2-K-NS) could induce in vitro chondrogenic differentiation and enhance the in vivo repair of damaged cartilage in an osteoarthritis model. In vitro, micromass cultures of murine and human stem cells with and without B2A were used as models of chondrogenic differentiation. Micromasses were evaluated for gene expression using microarray analysis and quantitative PCR; and for extracellular matrix production by Alcian blue staining for sulfated glycosaminoglycan and immunochemical detection of collagen type II. In vivo, osteoarthritis was chemically induced in knees of adult rats by an injection of mono-iodoacetate (MIA) into the synovial space. Treatment was administered at 7- and 14 days after the MIA by injection into the synovial space of B2A or saline and terminated at 21 days, after which knee cartilage damage was determined and scored by histological analysis. In murine C3H10T1/2 micromass culture, B2A induced the expression of more than 11 genes associated with growth factors/receptors, transcription, and the extracellular matrix, including PDGF-AA. B2A also significantly increased the sulfated glycosaminoglycan and collagen of murine- and human micromass cultures. In the knee osteoarthritis model, B2A treatment enhanced cartilage repair compared to untreated knees as determined histologically by a decrease in damage indicators. These findings suggest that B2A induces stem cells chondrogenic differentiation in vitro and enhances cartilage repair in vivo."

"B2A is a positive BMP-2 receptor modulator whose design is modeled on BMP and incorporates three domains: a heparin-binding domain, a hydrophobic domain, and a receptor-targeted domain. B2A-type peptides bind to type I and type II receptors, and appear to have a selectivity for BMPR-Ib{upregulated by LSJL}. B2A stimulation of cells increases ERK1/2 activation and, in the presence of BMP-2, augments Smads and alkaline phosphatase activities."

"Compared to the controls, 11 genes were found up-regulated in the B2A-treated micromass samples and one gene was down-regulated. Genes that were up-regulated in B2A treatment micromasses can be categorized into three groups: (1) genes associated with growth factors and growth factor receptors, (2) genes associated with transcription factors and gene regulation, and (3) genes of matrix proteins. In the first group, Fgfr1{upregulated in LSJL} and Fgfr2 were found significantly increased in the B2A-treated samples. We also detected a moderate up-regulation of growth factors Fgf1. In the second gene group, the Smad1, Smad4, and Twist1 were up-regulated. Twist1, it should be noted, is required to promote and maintain chondrogenic differentiation in immature chondrocytes. Among the matrix genes of the third gene group, collagens Col11a1{upregulated in LSJL} and Col3a1{upregulated in LSJL} were up-regulated, with Col3a1 being the most pronounced. In contrast, Col5a1, a collagen that is not associated with cartilage, was noticeably down-regulated by B2A. Phex and Serpinh1, genes that regulate extracellular matrix stability, were found to be increased. The Bmp1 gene, encoding a metalloproteinase, which induces ectopic cartilage formation, was found significantly increased in the B2A-treated cells. The microarray-base expression analyses also revealed that Sox9 and Col2 were up-regulated but did not reach statistical significance. In individual quantitative real-time PCR, Sox9 and Col2 were found increased in the B2A-treated micromasses with 1.3 (p < 0.05) and 1.6 (p < 0.0001) fold of control, respectively."

"PDGF-AA has been found to be a specific PDGF isoform associated with chondrogenic phenotype. It is a potent mitogenic and chemotactic factor for mesenchymal stem cells and chondrocytes, and can also increase proteoglycan production in chondrocytes. The increased Pdgfa might serve as an autocrine loop to propel the chondrogenic differentiation"

"Bmp1, a member of the BMP1/TLD metalloproteinases, was found significantly up-regulated (∼500 fold). In addition to playing critical roles in regulating the formation of various extracellular matrixes, BMP1 also regulates activity of TGF-β family members. BMP1 activates BMP2/4 by cleaving their antagonist, chordin. In vivo, BMP1 is able to induce ectopic cartilage."

Friday, September 4, 2009


Shox2 is required for chondrocyte proliferation and maturation in proximal limb skeleton.

"Mutations in the short stature homeobox gene SHOX lead to growth retardation associated with Turner, Leri-Weill dyschondrosteosis, and Langer mesomelic dysplasia syndromes, which marked the shortening of the forearms and lower legs. We report here that in contrast to the SHOX mutations in humans, Shox2 deficiency in mice leads to a virtual elimination of the stylopod in the developing limbs, while the zeugopod and autopod appear relatively normal. This phenotype is consistent with the restriction of the Shox2 expression to the proximal mesenchyme in the limb bud and later to chondrocytes associated with the forming stylopod. In the Shox2(-/-) embryo, the mesenchymal condensation for the stylopod initiates normally but the cartilaginous element subsequently fails in growth, chondrogenesis and endochondral ossification. A dramatic down-regulation of Runx2 and Runx3 could account for the lack of chondrocyte hypertrophy, while a down-regulation of Ihh expression may be responsible for a significant reduction in chondrocyte proliferation in the mutant stylopod. We further demonstrate that an enhanced and ectopic Bmp4 expression in the proximal limb of the Shox2 embryo may underlie the down-regulation of Runx2, as ectopically applied exogenous BMP4 represses Runx2 expression in the early limb bud. Moreover, we show that mouse Shox2, similar to human SHOX, can perform opposite roles on gene expression: either as a transcription activator or a repressor in different cell types. Our results establish a key role for Shox2 in regulating the growth of stylopod by controlling chondrocyte maturation via Runx2 and Runx3."

"deletions of Hoxa10, Hoxc10 and Hoxd10 {LSJL upregulates this} drastically shorten the femur, whereas loss of Hoxa11 and Hoxd11 results in a virtual loss of the radius and the ulna "

"Ectopic expression of either Meis1 or Meis2 in developing chick limbs reduces or truncates distal limb compartments"

"as limb bud elongates, Shox2 expression was confined to the mesenchyme of the proximal limb, with stronger expression remaining in the dorsal region"

Shox2 may affect Ihh expression.

The role of Shox2 in SAN development and function. states that Shox2 may have a role in Nkx3.2 repression.

Shox2 regulates progression through chondrogenesis in the mouse proximal limb.

" Prrx1{up in LSJL}-Cre-driven limb-specific deletion of the paralogous gene Shox2 results in severe rhizomelia. In this study, we show that Col2a1-Cre-driven deletion of Shox2 in developing chondrocytes also results in shortening of the stylopodial skeleton (i.e. humerus, femur) and that this rhizomelia is due to precocious chondrocyte maturation and hypertrophy. We demonstrate, using the micromass culture model system, that increased BMP activity triggers accelerated maturation and hypertrophy in Col2a1-Cre Shox2 mutant chondrocytes and we confirm in vivo that elevated transcript levels and expanded expression domains of Bmp2{up in LSJL} and 4 are associated with premature formation of the hypertrophic zone in mutant humeri. In micromass cultures of Prrx1-Cre Shox2 mutant limb cells, we find that Shox2 deletion in undifferentiated mesenchymal cells results in increased BMP activity that enhances early chondrogenesis, but is insufficient to provoke chondrocyte maturation and hypertrophy. Similarly, shRNA-mediated Shox2 knockdown in multipotent C3H10T1/2 cells and primary mouse bone marrow mesenchymal stem cells results in spontaneous chondrogenesis in the absence of chondrostimulation, but again fails to induce progression through the later stages of chondrogenic differentiation. Importantly, exogenous BMP supplementation can overcome the block to maturation and hypertrophy caused by Shox2 depletion prior to overt chondrogenesis. Thus, we provide evidence that Shox2 regulates progression through chondrogenesis at two distinct stages - the onset of early differentiation and the transition to maturation and hypertrophy."

"undifferentiated mesenchymal cells upregulate expression of the transcription factor Sox9{up} and commence chondrogenesis. Newly differentiated chondrocytes produce the characteristic cartilage extracellular matrix (ECM) components aggrecan (Acan){up} and collagen type II (Col2a1){up} and proliferate to expand the size of the nascent cartilage anlage.  Prompted by Runx2 expression, proliferating chondrocytes at the center of the template exit the cell cycle and initiate hypertrophic differentiation. Hypertrophic chondrocytes express the unique marker collagen type X (Col10a1){up} and experience a massive increase in cytoplasmic volume. Perichondrial cells and chondrocytes at the ends of the cartilage template express Pthlp, which stimulates chondrocytes to proliferate. As these proliferating growth plate chondrocytes leave the domain of PTHLP influence, they mature into prehypertrophic chondrocytes and express Ihh. It is the enormous increase in hypertrophic chondrocyte volume and the columns of rapidly proliferating chondrocytes that are responsible for driving longitudinal bone growth. Finally, hypertrophic chondrocytes express genes like Bglap, Ibsp, and Spp1 in order to mineralize their ECM before undergoing apoptosis"

"Col2a1-Cre Shox2 mutant cultures express significantly lower levels of genes involved in early chondrogenesis, like Sox9, Sox6, Acan, and Col2a1, and chondrocyte maturation, like Runx2 and Ihh, but significantly higher levels of the hypertrophic marker Col10a1"

"chondrocyte-specific Shox2 deletion significantly increased levels of Bmp2 and 4 mRNAs"

"Prrx1-Cre Shox2 mutant cultures express significantly lower levels of the tenogenic marker gene Scx{up} and the myogenic markers Myf5, Myod1, and Myog{down}"

"when embryonic chick limb bud cells are cultured in micromass, limb positional information is gradually lost unless the cells are exposed to PD patterning signals, like retinoic acid (RA), FGF8, and WNT3A"

"mutant micromasses with 10 ng/ml Noggin for the last 3 days of a 7-day culture period and found that Noggin completely blocked the internodular chondrogenesis normally observed in Prrx1-Cre Shox2 mutant cultures"<-Could Shox2 deletion induce ectopic chondrogenesis?

"Col2a1-Cre-driven conditional Shox2 deletion causes significant shortening of the humerus and femur and that this rhizomelia is caused by precocious hypertrophic differentiation of stylopodial chondrocytes"

"Shox2 knockdown induces MSC chondrogenesis without hypertrophy, as long as Shox2-deficient MSC pellets are kept BMP-free"