Monday, October 12, 2009


Gradients in bone morphogenetic protein-related gene expression across the growth plate.

"In the growth plate, stem-like cells in the resting zone differentiate into rapidly dividing chondrocytes of the proliferative zone and then terminally differentiate into the non-dividing chondrocytes of the hypertrophic zone. To explore the molecular switches responsible for this two-step differentiation program, we developed a microdissection method to isolate RNA from the resting (RZ), proliferative (PZ), and hypertrophic zones (HZ) of 7-day-old male rats. Expression of approximately 29,000 genes was analyzed by microarray and selected genes verified by real-time PCR. The analysis identified genes whose expression changed dramatically during the differentiation program, including multiple genes functionally related to bone morphogenetic proteins (BMPs). BMP-2 and BMP-6 were upregulated in HZ compared with RZ and PZ (30-fold each, P < 0.01 and 0.001 respectively). In contrast, BMP signaling inhibitors were expressed early in the differentiation pathway; BMP-3 and gremlin were differentially expressed in RZ (100- and 80-fold, compared with PZ, P < 0.001 and 0.005 respectively) and growth differentiation factor (GDF)-10 in PZ (160-fold compared with HZ, P < 0.001). Our findings suggest a BMP signaling gradient across the growth plate, which is established by differential expression of multiple BMPs and BMP inhibitors in specific zones. Since BMPs can stimulate both proliferation and hypertrophic differentiation of growth plate chondrocytes, these findings suggest that low levels of BMP signaling in the resting zone may help maintain these cells in a quiescent state. In the lower RZ, greater BMP signaling may help induce differentiation to proliferative chondrocytes. Farther down the growth plate, even greater BMP signaling may help induce hypertrophic differentiation."

"Mice deficient in both BMP receptor-1a and -1b in cartilage lack the majority of skeletal elements that form through endochondral ossification"<-LSJL upregulates BMPR1B.

"Inhibitors of BMP signaling, BMP-3, GDF-10, gremlin, and chordin were expressed early in the differentiation pathway."

"BMPR1a was detected at similar levels throughout the growth plate, whereas BMPR-1b was found to be expressed approximately eightfold higher in the HZ than in the PZ "

"In the growth plate, we found that BMP-2 and -6 are expressed primarily in the hypertrophic zone at levels similar to or greater than those found in the trabecular bone of the metaphysis. In contrast, BMP-7, previously shown to inhibit growth plate chondrocyte differentiation in cultured fetal metatarsal bones, was found to be expressed at the highest levels in PZ."

"treatment with Noggin, a BMP antagonist, inhibits proliferation of resting zone chondrocytes and hypertrophy of proliferative zone chondrocytes"

"Overexpression of a constitutively active BMPR1a in mice accelerates hypertrophic differentiation, but has no effect on proliferation"

"BMPR1a may mediate the effects of BMPs earlier in the differentiation pathway, especially in the transition from resting to proliferative phenotype, whereas BMPR1b may preferentially mediate the effects of BMPs on hypertrophic differentiation."

"BMP agonists were expressed primarily in HZ, whereas BMP antagonists were expressed primarily in the resting zone."

"The objective of this research was to detect bovine GDF10 gene polymorphism and analyze its association with body measurement traits (BMT) of animals sampled from 6 different Chinese indigenous cattle populations. The populations included Xuelong (Xl), Luxi (Lx), Qinchuan (Qc), Jiaxian red (Jx), Xianang (Xn) and Nanyang (Ny). Blood samples were taken from a total of 417 female animals stratified into age categories of 12-36 months. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was employed to find out GDF10 single polymorphism nucleotide (SNPs) and explore their possible association with BMT. Sequence analysis of GDF10 gene revealed 3 SNPs in total: 1 in exon1 (G142A) and 2 in exon3 (A11471G, and T12495C). G142A and T12495C SNPs are both synonymous mutation. They showed 2 genotypes namely respectively (GG, GA) and (PP and PB). A11471G SNP is a missense mutation leading to the change of Alanine to Threonine amino acid. It showed three genotypes namely AA, BB and AB. Analysis of association of polymorphism with body measurement traits at the three locus showed that there were significant effects on BMT in Qc, Jx and Ny cattle population. These results suggest that the GDF10 gene might have potential effects on body measurement traits in the above mentioned cattle populations and could be used for marker-assisted selection."

Body Length(BL)
Height at Hips(HH)

A PB mutation at exon 3 increased body length.

"GDF10 has been discovered in rat and human femur tissue"

"BMP4 and GDF9 were found to be associated with litter size in goat "

Bone morphogenetic protein-3b (BMP-3b) inhibits osteoblast differentiation via Smad2/3 pathway by counteracting Smad1/5/8 signaling.

"Despite the involvement of BMP-3b (also called GDF-10) in osteogenesis, embryogenesis and adipogenesis, the functional receptors and intracellular signaling of BMP-3b have yet to be elucidated. In the present study, we investigated the cellular mechanism of BMP-3b in osteoblast differentiation using mouse myoblastic C2C12 cells. BMP-3b stimulated activin/TGF-β-responsive promoter activities. The stimulatory actions of BMP-3b on activin/TGF-β-responsive activities were suppressed by co-treatment with BMP-2. BMP-responsive promoter activities stimulated by BMP-2 were significantly inhibited by treatment with BMP-3b. BMP-3b suppressed the expression of osteoblastic markers including Runx2, osteocalcin and type-1 collagen induced by BMP-2, -4, -6 and -7. BMP-2-induced Smad1/5/8 phosphorylation and mRNA levels of the BMP target gene Id-1 were suppressed by co-treatment with BMP-3b, although BMP-3b failed to activate Smad1/5/8 signaling. Of interest, the BMP-3b suppression of BMP-2-induced Id-1 expression was not observed in cells overexpressing Smad4 molecules. On the other hand, BMP-3b directly activated Smad2/3 phosphorylation and activin/TGF-β target gene PAI-1 mRNA expression, while BMP-2 suppressed BMP-3b-induced Smad2/3 signal activation. BMP-2 inhibition of BMP-3b-induced PAI-1 expression was also reversed by overexpression of Smad4. Analysis using inhibitors for BMP-Smad1/5/8 pathways revealed that these BMP-3b effects were mediated via receptors other than ALK-2, -3 and -6. Furthermore, results of inhibitory studies using extracellular domains for BMP receptor constructs showed that the activity of BMP-3b was functionally facilitated by a combination of ALK-4 and ActRIIA. Collectively, BMP-3b plays an inhibitory role in the process of osteoblast differentiation, in which BMP-3b and BMP-2 are mutually antagonistic possibly by competing with the availability of Smad4."

"Sca-1, a stem cell marker having a regenerative capacity, maintains the growth and invasive characteristics of tumor cells in part by suppressing the expression of BMP-3b, leading to inhibition of TGF-β signaling through BMP-3b"

BMP-6 and BMPR-1a are up-regulated in the growth plate of the fractured tibia.

In the joint loading study, the non-loaded limb was compared to the loaded limb so it's possible that BMP-6 and BMPR-1a were upregulated in both limbs thus accounting for the growth increase in the contralateral versus control limbs.

"Bone overgrowth is a known phenomenon occurring after fracture of growing long bones with possible long-term physical consequences for affected children. Here, the physeal expression of bone morphogenetic proteins (BMPs) was investigated in a fracture-animal model to test the hypothesis that a diaphyseal fracture stimulates the physeal expression of these known key regulators of bone formation, thus stimulating bone overgrowth. Sprague-Dawley rats (male, 4 weeks old), were subjected to a unilateral mid-diaphyseal tibial fracture. Kinetic expression of physeal BMP-2, -4, -6, -7, and BMP receptor-1a (BMPR-1a) was analyzed in a monthly period by quantitative real time-polymerase chain reaction and immunohistochemistry. On Days 1, 3, 10, and 14 post-fracture, no changes in physeal BMPs gene-expression were detected. Twenty-nine days post-fracture, when the fracture was consolidated, physeal expression of BMP-6 and BMPR-1a was significantly upregulated in the growth plate of the fractured and contra-lateral intact bone compared to control (p < 0.005)[or it could be that the duration was not long enough, gene expression after joint loading was done after 49 hours but an increase in contralateral bone length was still observed after studies taking place only 10 days]. This study demonstrates a late role of BMP-6 and BMPR-1a in fracture-induced physeal growth alterations and furthermore, may have discovered the existence of a regulatory "cross-talk" mechanism between the lower limbs whose function could be to limit leg-length-discrepancies following the breakage of growing bones. "

"An average overgrowth following shaft fracture of the femur has been defined to be almost 1 cm (range 0.4–2.7 cm) in a review of 74 patients under the age of 13."

They also detected a change in BMP-7.

"Postnatally, [BMPs] are expressed at the physis in resting (BMP-3), proliferating (BMP-7) and pre-/hypertrophic chondrocytes (BMP-2 and -6), as well as in the perichondrium and periosteum (BMP-2, -3, -4, -5, and -7)."

" BMP-6, -7, and -receptor 1a showed a tendency towards a lower expression in physeal tissue of broken bones compared to the contra-lateral and the control group during the acute inflammatory phase of fracture healing (Day 1 and Day 3). BMP-4 mRNA levels were lowered only on Day 1 compared to control animals and to the contra-lateral non-fractured bon. Ten and 14 days after fracture, no significant differences in physeal gene-expression could be observed between the different groups. Thus, as no significant differential physeal expression of BMP-2, -4, -6, -7, and BMPR-1a was observed during inflammatory, fibrogenic, and early osteogenic stages of fracture healing, our results indicate that BMP-2, -4, -6, and -7 do not account for an enhanced proliferation rate of physeal chondrocytes, nor an up-regulation of mitogenic genes seen shortly after fracture."

"apoptosis rates of physeal chondrocytes achieved their maximum on Day 29 post diaphyseal fracture."

"BMP-6 and BMPR-1a were deregulated in both the broken and contra-lateral non-broken tibiae 29 days post-fracture"

"BMP-6, redundantly expressed by hypertrophic chondrocytes, contributes to posttraumatic overgrowth by exerting its stimulating effect on chondrogenesis and osteogenesis via BMPR-1a activation during later stages of bone healing."

I sent a comment to the author that contralateral overgrowth was induced in LSJL studies before day 29 and that no evidence was seen of an upregulation of BMPR-1a and BMP-6 in the LSJL studies however it's possible that these two BMP's were upregulated in both limbs thus canceling each other out.

Here was her response:

"I am aware that tensile dynamic loading applied to growing bones stimulates growth and therefore might be used to overcome leg length discrepancies..."

Although more loading is involved in LSJL than tensile dynamic loading like hydrostatic pressure and fluid flow.

"Our paper however addressed to analyze the input of a diaphyseal fracture on the nearby growth plate and showed that BMP -6 and the revozier 1a are upregulated at later stages of fracture healing which was also observed at the contralateral side on day 29 but not at the earlier investigated time-points, with the last one before, being day 14th.
I don't think that it is possible to compare our results with a joint loading study, as the fractured bone in our study was not loaded at all during early stages of feacture healing due to instable fracture conditions.

Nevertheless, I think, that we can draw the connection in the context that the contralateral side does respond to any kind of trauma or external stimulus, eventhough the bone is kept untouched in the experiments.
Bmp -6 seems to be one target gene of the whole process which certainly involves several signalling pathways including systemic growth hormons."

BMPs regulate multiple aspects of growth-plate chondrogenesis through opposing actions on FGF pathways.

"we characterized the skeletal phenotypes of mice lacking Bmpr1a in chondrocytes (Bmpr1a(CKO)) and Bmpr1a(CKO);Bmpr1b+/- (Bmpr1a(CKO);1b+/-) in order to test the roles of BMP pathways in the growth plate in vivo. These mice reveal requirements for BMP signaling in multiple aspects of chondrogenesis. The balance between signaling outputs from BMP and fibroblast growth factor (FGF) pathways plays a crucial role in the growth plate. BMP signaling is required to promote Ihh expression, and to inhibit activation of STAT and ERK1/2 MAPK, key effectors of FGF signaling. BMP pathways inhibit FGF signaling, at least in part, by inhibiting the expression of FGFR1."

"there was a significant increase in activated STAT1- and STAT5a-positive chondrocytes in the proliferative zones in Bmpr1aCKO;1b+/- mice"

"FGFR1 levels are increased in Bmpr1aCKO and Bmpr1aCKO;1b+/- growth plates "

"the Ihh promoter is responsive to SMAD1 in chondrocytes, suggesting that BMPs induce Ihh, and that this regulation may be direct. "

"BMP signaling partially inhibits ERK1/2 MAPK."

No comments:

Post a Comment