Wednesday, March 25, 2009

BJIG

Effects of Bojungikgitang-gagambang on Longitudinal Bone Growth in Adolescent Rats

"This study was aimed to investigate the effectof Bojungikgitang-gagambang (BJIG) on longitudinal bone growth in rats.
The BJIG treated group (300 mg/kg) and the control group (vehicle) were administered orally twice daily for 4 days. To investigate the effects of BJIG we measured body weight gain. The bone growth effect was analyzed by measuring between fluorescent lines marked with tetracycline, which plays the role of fluorescent dye on the surface of the tibia. Tetracycline was intraperitoneally injected. The height of growth plates in the epiphyseal plate was measured. The expression of bone morphogenetic protein-2 (BMP-2) and insuline-like growth factor-1 (IGF-1) was investigated by immunohistochemistry.
BJIG caused a significant acceleration of longitudinal bone growth of 349.7 ± 15.9 /day compared to control (319.8 ± 21.4 /day). The height of overall growth plate was not significantly more compared to the control, but the size of cells in the proliferative zone and hypertrophic zone were. In the immunohistochemistry, BMP-2 and IGF-1 were expressed markedly in the proliferative or hypertrophic zone, respectively.
BJIG stimulated the chondrocyte hypertrophy and chondrogenesis in the growth plate and directly increased the longitudinal tibia length of rats."

It's a foreign language study so couldn't get the full version.  Also I could not find the ingredients.

Wednesday, March 18, 2009

c-Jun

c-Jun is upregulated by LSJL.

c-Jun is required for the specification of joint cell fates.

"c-Jun [is] a candidate transcription factor activating [the] Wnt9a enhancer element. c-Jun is specifically expressed in joints during embryonic joint development, and its conditional deletion from early limb bud mesenchyme in mice severely affects both initiation and subsequent differentiation of all limb joints. c-Jun directly regulates Wnt16 as well as Wnt9a during early stages of joint development, causing a decrease of canonical Wnt activity in the joint interzone. Postnatally, c-Jun-deficient mice show a range of joint abnormalities, including cartilaginous continuities between juxtaposed skeletal elements, irregular articular surfaces, and hypoplasia of ligaments."

"Wnt9a misexpression can induce expression of a wide range of joint markers, including Gdf5, Autotaxin, and Chordin, in chondrogenic regions"

"gain of function of Gdf5 does not result in joint formation in the developing limb "

"verexpression of c-Jun enhanced endogenous Wnt9a expression, whereas that of a truncated form of c-Jun, which lacks its N-terminal transactivation domain (c-Jun ΔN, also known as Tam67), suppressed the Wnt9a expression "


Figure 4.
"Deletion of c-Jun from early mesenchyme causes cartilaginous continuity between juxtaposed skeletal elements. (A) Alcian blue staining on sections of interphalangeal joints in c-Jun mutants and control mice (E14.5 and E16.5). A red arrowhead indicates a joint interzone, characterized by condensed localized cells in control mice. Bars, 100 μm. (B) Skeletal staining of interphalangeal and knee joints of c-Jun mutants and control mice at P0. Red arrowheads indicate the absence of Alcian blue staining at joint spaces in control mice. Bars, 500 μm. (C) Alcian blue staining on sections of the knee joints of c-Jun mutants and control mice at E16.5 and P0. Intercondylar space and articulation between femoral condyle and tibial plateau are shown separately. Green arrows show cruciate ligaments in control mice, which are not visible in c-Jun mutants. Boxed areas are magnified on the right. Green lines on the side indicate a joint cavity space where ligaments normally arise. A solid red arrowhead shows articular cartilage layer at E16.5, characterized by condensed cell layers with a decrease in Alcian blue staining. The layer is more evident at P0 (open red arrowhead). A blue line indicates cartilage condensation in the tibia. Bars, 100 μm. "

So C-Jun KO can results in ectopic chondrogenesis but probably not height due to the dysregulation caused.

"JunB, JunD, Fra2, and Atf2 are weakly expressed in developing joints and were also found at a comparable level in the surrounding perichondrium, while other AP1 family molecules (c-Fos, FosB, and Fra1) were not detectable in developing joints"

"We found a dramatically decreased level of Axin2, a classical Wnt canonical target, in the developing joints of c-Jun mutants."

"-Jun mutants show the abnormal formation of joint interzone molecularly and morphologically, including failure to express several interzone-specific genes, such as Autotaxin and Chordin"

Saturday, March 14, 2009

Salubrinal

 Hiroki Yokota participated in this study.

Salubrinal Reduces Expression and Activity of MMP13 in Chondrocytes.

"Stress to the endoplasmic reticulum (ER) and inflammatory cytokines induce expression and activity of matrix metalloproteinase 13 (MMP13). Since a synthetic agent, salubrinal, is known to alleviate ER stress and attenuate nuclear factor kappa B (NFκB) signaling, we addressed a question whether upregulation of MMP13 by ER stress and cytokines is suppressed by administration of salubrinal.
Using C28/I2 human chondrocytes, we applied ER stress with tunicamycin and inflammatory distress with tumor necrosis factor α (TNFα) and interleukin 1β (IL1β). RNA interference with siRNA specific to NFκB p65 (RelA) was employed to examine a potential involvement of NFκB signaling in salubrinal's action in regulation of MMP13. We also employed primary human chondrocytes and evaluated MMP13 activity.
Tunicamycin activated p38 MAPK, while inflammatory cytokines activated p38 MAPK and NFκB. In both cases, salubrinal significantly reduced expression and activity of MMP13. Silencing NFκB reduced inflammatory cytokine-driven upregulation of MMP13 activity.
Salubrinal downregulates expression and activity MMP13 through p38 and NFκB signaling."

Now MMP13 is actually essentially for endochondral ossification.

"elevation of the level of phosphorylated eIF2a leads to expression of activating transcription factor 4 (ATF4)"<-salubrinal inhibits EIF2a dephosphorylation.

"In response to 10 ng/ml IL1b and 1 mg/ml tunicamycin, the mRNA levels of MMP1, MMP2{up}, and MMP14{up} were not altered, but the level of MMP3{up} mRNA was elevated"

"salubrinal decreased the level of p-IKK that was known to downregulate the NFkB inhibitor, IkB"

This study does present a clear linkage between Salubrinal and height but doesn't rule out increase applications.

"Tunicamycin is an inhibitor of N-linked glycosylation and the formation of N-glycosidic protein-carbohydrate linkages"

"Tunicamycin increased the level of eIF2a phosphorylation (p-eIF2a) at 1 h, followed by an elevation in the level of ATF4 at 3 h"

"Co-incubation with tunicamycin and salubrinal presented an increase in p-eIF2a and ATF4"

"Tunicamycin induced phosphorylation of p38 MAPK (p-p38 MAPK) at 1 and 2 h, while administration of 10 mM salubrinal suppressed the tunicamycin-induced increase in p-p38 MAPK"  Tunicaymin did not activate NFkB.

The following article is by the LSJL lab:

Effects of salubrinal on development of osteoclasts and osteoblasts from bone marrow-derived cells.

"Osteoporosis is a skeletal disease leading to an increased risk of bone fracture. Using a mouse osteoporosis model induced by administration of a receptor activator of nuclear factor kappa-B ligand (RANKL), salubrinal was recently reported as a potential therapeutic agent. To evaluate the role of salubrinal in cellular fates as well as migratory and adhesive functions of osteoclast/osteoblast precursors, we examined the development of primary bone marrow-derived cells in the presence and absence of salubrinal{so we can see any potential effects of BM-MSCs on salubrinal for LSJL}.
Using the RANKL-injected and control mice, bone marrow-derived cells were harvested. Osteoclastogenesis was induced by macrophage-colony stimulating factor and RANKL, while osteoblastogenesis was driven by dexamethasone, ascorbic acid, and beta-glycerophosphate.
Salubrinal suppressed the numbers of colony forming-unit (CFU)-granulocyte/macrophages and CFU-macrophages, as well as formation of mature osteoclasts in a dosage-dependent manner. Salubrinal also suppressed migration and adhesion of pre-osteoclasts and increased the number of CFU-osteoblasts. Salubrinal was more effective in exerting its effects in the cells isolated from the RANKL-injected mice than the control. Consistent with cellular fates and functions, salubrinal reduced the expression of nuclear factor of activated T cells c1 (NFATc1) as well as tartrate-resistant acid phosphatase.
Salubrinal exhibits significant inhibition of osteoclastogenesis as well as stimulation of osteoblastogenesis in bone marrow-derived cells, and its efficacy is enhanced in the cells harvested from the osteoporotic bone samples."

We need of Salubrinal's effects on MSC's pre-differentiation to see if it can have an impact on LSJL.

"Salubrinal is reported to attenuate molecular signaling mediated by nuclear factor kappa B (NFκB)"

"C57BL/6 female mice (7 weeks of age) were used"

"mRNA and  protein expression of NFATc1 is downregulated by salubrinal. NFATc1 is a member of the
NFAT transcription factor family and a master transcription factor for osteoclast development. NFATc1-deficient embryonic stem cells are unable to differentiate into osteoclasts"

"MafB (V-maf musculoaponeurotic fibrosarcoma oncogene homolog B), IRF8 (interferon regulatory factor
8), and Bcl6 (V cell lymphoma) have been mentioned as inhibitors of NFATc1"<-Salubrinal may act through these inhibitors and if it does in turn the effects of these inhibitors could have importance for LSJL.

Wednesday, March 11, 2009

Platelet-Rich Plasma

Platelet-Rich Plasma for the Replenishment of Bone

"Platelet-rich plasma (PRP) is the term applied to blood plasma enriched with platelets"

"Platelet-rich plasma (PRP) contains transforming growth factor-β (TGF-β) that attracts osteoblast progenitors to sites needing new bone formation. PRP is produced from anticoagulated whole blood by removing red blood cells (RBCs) and leukocytes, and concentrating platelets in the product. The resulting plasma contains platelets, which when activated release significant amounts of platelet-derived growth factor (PDGF) and TGF-β. After injection of PRP into sites of skeletal injury, the PRP serves as a source to locally increase the amounts of PDGF and TGF-β. It is thought that these factors recruit bone marrow stromal cells"<-can this TGF-B and recruitment of MSCs induce ectopic growth plates as well?

"PRP contains an angiogenesis inhibitor, thrombospondin-1 (TSP-1)."

"Osteoblast-specific overexpression of TGF-β in mice causes excess osteoclast activation leading to high turnover bone loss"<-could this excess osteoclast activation allow for new growth plate formation?

TGF-B1 is released by osteoclastic resorption.

Platelet-Rich Plasma in the Pathologic Processes of Cartilage: Review of Basic Science Evidence.

"The effects of PRP [include] increasing chondrocyte and mesenchymal stem cell proliferation, proteoglycan deposition, and type II collagen deposition. PRP was also found to increase the cell viability of chondrocytes and the migration and chondrogenic differentiation of mesenchymal stem cells (MSCs) and to inhibit the effect of catabolic cytokines."

When Human Mesenchymal Stem Cells were cultured in 10% plasma both osteogenic and chondrogenic genes increased with chondrogenic genes being increased at a greater level.

Saturday, March 7, 2009

Quinoline

Benzofuro[3,2-c]quinoline compounds

General information on the formula is stated on the linked page and also that it has a stimulatory effect on longitudinal bone growth.

The maximum potency for the effect on femoral length was 1.10.

Mustn1

Mustn1 is expressed during chondrogenesis and is necessary for chondrocyte proliferation and differentiation in vitro.

"Mustn1 encodes a small nuclear protein expressed specifically in the musculoskeletal system that was originally identified as a strongly up-regulated gene during bone regeneration, especially in fracture callus proliferating chondrocytes. Further experiments were undertaken to investigate its expression and role during chondrogenesis. Initially, whole mount mouse in situ hybridization was carried out and revealed Mustn1 expression in areas of active chondrogenesis that included limb buds, branchial arches and tail bud. To elucidate its function, experiments were carried out to perturb Mustn1 by overexpression and silencing in the pre-chondrocytic RCJ3.1C5.18 (RCJ) cell line. In these cells, Mustn1 is normally differentially regulated, with a spike in expression 2 days after induction of differentiation. Further, Mustn1 was successfully overexpressed in multiple RCJ cell lines by approximately 2-6 fold, and reduced to approximately 32-52% in silenced cell lines as compared to parental Mustn1 levels. Overexpressing, silenced, control, and parental RCJ cell lines were assayed for proliferation and differentiation. No statistically significant changes were observed in either proliferation or proteoglycan production when Mustn1 overexpressing lines were compared to parental and control{so optimal Mustn1 is likely necessary for height growth but excess quantities won't result in extra height}. By contrast, both proliferation rate and differentiation were significantly reduced in Mustn1 silenced cell lines. Specifically, RNAi silenced cell lines showed reductions in populations of approximately 55-75%, and also approximately 34-40% less matrix (proteoglycan) production as compared to parental and random control lines. Further, this reduction in matrix production was accompanied by significant downregulation of chondrogenic marker genes, such as Sox9, Collagen type II (Col II), and Collagen type X (Col X). Lastly, reintroduction of Mustn1 into a silenced cell line rescued this phenotype, returning proliferation rate, matrix production, and chondrogenic marker gene expression back to parental levels."

Fracture vs. LSJL gene expression

Differential gene expression analysis in fracture callus of patients with regular and failed bone healing.

"Between March 2006 and May 2007, a total of 130 patients who were surgically treated at the Berufsgenossenschaftliche Unfallklink Ludwigshafen were screened for the study. Tissue samples of patients with normal and failed fracture healing were collected intraoperatively. Patients were divided into groups depending on the fracture date, and only patients with fractures two to four weeks old and patients with non-unions more than 9 months old were included in the final analysis."

Individuals from 18-80 were used.

"In chondrogenesis, fibronectin is thought to have a significant role in the differentiation of mesenchymal cells to chondral cells."

"The cytoskeletal gene ACTA2, which encodes the human aortic smooth muscle actin, is more than two fold higher expressed in tissue samples of patients with non-unions. Actin is the major component of microfilaments, and plays an important role in maintaining cell shape and movement. Smooth muscle α-actin is expressed in vascular smooth muscle cells and fibroblasts."

Genes upregulated in non-union fractures versus union fractures also upregulated in LSJL:

Acta2

Loss of transcription factor early growth response gene 1 results in impaired endochondral bone repair.

"Here, we assessed the consequence of loss of early growth response gene 1 (EGR-1){up} on endochondral bone healing because this transcription factor has been shown to modulate repair in vascularized tissues. Model fractures were created in ribs of wild type (wt) and EGR-1(-/-) mice. Differences in tissue morphology and composition between these two animal groups were followed over 28 post fracture days (PFDs). In wt mice, bone healing occurred in healing phases characteristic of endochondral bone repair. A similar healing sequence was observed in EGR-1(-/-) mice but was impaired by alterations. A persistent accumulation of fibrin between the disconnected bones was observed on PFD7 and remained pronounced in the callus on PFD14. Additionally, the PFD14 callus was abnormally enlarged and showed increased deposition of mineralized tissue. Cartilage ossification in the callus was associated with hyper-vascularity and -proliferation. Moreover, cell deposits located in proximity to the callus within skeletal muscle were detected on PFD14. Despite these impairments, repair in EGR-1(-/-) callus advanced on PFD28, suggesting EGR-1 is not essential for healing."

"A majority of bone marrow cells expressed EGR-1 [in the fracture callus], while no EGR-1 was detected in the bone-embedded osteocytes"

"Cartilage formation in EGR-1−/− callus extended frequently perpendicular to the periosteal bone, while cartilage in wt callus formed adjacent to the bone surface"

"The EGR-1−/− mouse callus showed a dumbbell-shaped, enlarged area of ossification"

EGR-1-/- had cartilage deposits within the muscle.

"expression of both PDGF-A/B and fibroblast growth factor 2 is positively regulated by EGR-1"

The biology of fracture healing.

Minor motion is good for fracture healing whereas too much delays fracture healing.

"Immediately following the trauma, a hematoma is generated and consists of cells from both peripheral and intramedullary blood, as well as bone marrow cells. The injury initiates an inflammatory response which is necessary for the healing to progress{microfracture may not induce an inflammatory response thus can it induce the healing process?}. The response causes the hematoma to coagulate in between and around the fracture ends, and within the medulla forming a template for callus formation."

"The initial proinflammatory response involves secretion of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6{up}, IL-11 and IL-18."

"The TNF-α concentration has been shown to peak at 24h and to return to baseline within 72h post trauma."

"TNFR1 is always expressed in bone whereas TNFR2 is only expressed following injury"

"IL-1 and IL-6 are believed to be most important for fracture healing"<-so the upregulation of IL-6 may be sufficient.

"TGF-β2, -β3 and GDF-5 are involved in chondrogenesis and endochondral ossification, whereas BMP-5 and -6 have been suggested to induce cell proliferation in intramembranous ossification at periosteal sites."

"Bone remodelling has been shown to be a result of production of electrical polarity created when pressure is applied in a crystalline environment. This is achieved when axial loading of long bones occur, creating one electropositive convex surface, and one electronegative concave surface, activating osteoclastic and osteoblastic activity respectively. By these actions the external callus is gradually replaced by a lamellar bone structure, whereas the internal callus remodelling re-establishes a medullar cavity characteristic of a diaphyseal bone."

"Bone on one side of the cortex must unite with bone on the other side of the cortex to re-establish mechanical continuity. If the gap between bone ends is less than 0.01 mm and interfragmentary strain is less than 2%, the fracture unite by so-called contact healing. Under these conditions, cutting cones are formed at the ends of the osteons closest to the fracture site. The tips of the cutting cones consist of osteoclasts which cross the fracture line, generating longitudinal cavities at a rate of 50–100 μm/day. These cavities are later filled by bone produced by osteoblasts residing at the rear of the cutting cone. This results in the simultaneous generation of a bony union and the restoration of Haversian systems formed in an axial direction. The re-established Haversian systems allow for penetration of blood vessels carrying osteoblastic precursors. The bridging osteons later mature by direct remodelling into lamellar bone resulting in fracture healing without the formation of periosteal callus."

"Gap healing differs from contact healing in that bony union and Haversian remodelling do not occur simultaneously. It occurs if stable conditions and an anatomical reduction are achieved, although the gap must be less than 800 μm to 1 mm. In this process the fracture site is primarily filled by lamellar bone oriented perpendicular to the long axis, requiring a secondary osteonal reconstruction unlike the process of contact healing. The primary bone structure is then gradually replaced by longitudinal revascularized osteons carrying osteoprogenitor cells which differentiate into osteoblasts and produce lamellar bone on each surface of the gap. This lamellar bone, however, is laid down perpendicular to the long axis and is mechanically weak. This initial process takes approximately 3 and 8 weeks, after which a secondary remodelling resembling the contact healing cascade with cutting cones takes place. Although not as extensive as endochondral remodelling, this phase is necessary in order to fully restore the anatomical and biomechanical properties of the bone"

"The acute inflammatory response peaks within the first 24h and is complete after 7 days"

Upregulation of inflammatory genes and downregulation of sclerostin gene expression are key elements in the early phase of fragility fracture healing.

"Fifty-six patients submitted to hip replacement surgery after a low-energy hip fracture were enrolled in this study. The patients were grouped according to the time interval between fracture and surgery: bone collected within 3 days after fracture (n = 13); between the 4(th) and 7(th) day (n = 33); and after one week from the fracture (n = 10). Inflammation- and bone metabolism-related genes were assessed at the fracture site. The expression of pro-inflammatory cytokines was increased in the first days after fracture. The genes responsible for bone formation and resorption were upregulated one week after fracture. The increase in RANKL expression occurred just before that, between the 4(th)-7(th) days after fracture. Sclerostin expression diminished during the first days after fracture.
The expression of inflammation-related genes, especially IL-6, is highest at the very first days after fracture but from day 4 onwards there is a shift towards bone remodeling genes, suggesting that the inflammatory phase triggers bone healing. We propose that an initial inflammatory stimulus and a decrease in sclerostin-related effects are the key components in fracture healing. In osteoporotic patients, cellular machinery seems to adequately react to the inflammatory stimulus, therefore local promotion of these events might constitute a promising medical intervention to accelerate fracture healing."

Genetic factors responsible for long bone fractures non-union.

"We carried out studies in patients with delayed long bone fractures estimating the frequency of mutation of genes crucial for pathogen recognition (TLR2, TLR4 and CD14), and elimination (CRP, IL-6, IL-1ra), as well as wound healing (TGF-β). The molecular milieu regulating healing process (IGF-1, COLL1a, TGF-β, BMP-2, and PDGF) was validated by Western blot analysis of the gap tissue.
Microbiological investigations showed the presence of viable bacterial strains in 34 out of 108 gaps in patients with non-healing fractures (31.5%) and in 20 out of 122 patients with uneventful healing (16.4%). The occurrence of mutated TLR4 1/W but not 2/W gene was significantly higher in the non-healing infected than sterile group. In the non-healing infected group 1/W mutated gene frequency was also higher than in healing infected. In the TGF-β codon 10 a significantly higher frequency of mutated homozygote T and heterozygote C/T in the non-healing infected versus non-healing sterile subgroup was observed. Similar difference was observed in the non-healing infected versus healing infected subgroup. The CRP (G1059C), IL1ra (genotype 2/2), IL-6 (G176C), CD14 (G-159T), TLR2 (G2259A) and TLR4/2 (Thr399Ile) polymorphisms did not play evident role in the delay of fracture healing.
Individuals bearing the mutant TLR 4 gene 1/W (Asp299Gly) and TGF-β gene codon 10 mutant T and T/C allele may predispose to impaired pathogen recognition and elimination, leading to prolonged pathogen existence in the fracture gaps and healing delays."

Wednesday, March 4, 2009

Height Growth with You Gui Yin?

It is available on Amazon: Astra Essence(Restorative Herbal Supplement, Zuo Gui Wan/You Gui Yin)(90 tabs) HC-11.   Also known as Restore the Right Kidney Detoction.

[Progressive studies on effects of traditional Chinese medicines on differentiation of human bone mesenchymal stem cells]. states "youguiyin-containing rabbit serum can induce BMSC differentiate into chondrocyte".  I haven't found the full study yet.

According to [Research of You Gui Yin and MSCs interventional therapy on early avascular necrosis of the femoral head]., You Gui Yin can also increase VEGF levels.

[Effects of different concentrations of gubishu containing serum on the proliferation of rabbit articular chondrocytes in vitro culture].

"Articular chondrocytes were obtained from the cartilage of 1-month rabbit and cultured in vitro. They were randomly divided into 8 groups,blank and Gubishu groups in different concentrations (5%, 10%,15%, 20%), MTT assay method was adopted to observe the influence of Gubishu containing serum with different concentrations to the proliferation of chondrocytes after incubated 1, 3, 5, 7 and 9 days.
The proliferation of chondrocytes was dependent on the concentration in Gubishu groups. At same time point,there was significant value between every groups, 20% concentration was greatest (P<0.05); There was significant differences between 5%, 10% and 20% concentration of the blank groups at same time point (P<0.05), and was not between 15% and 20% concentration at the 1, 3, 5 and 7 days (P>0.05), 20% concentration of the blank group was greatest. 20% concentrations of Gubishu containing serum was significantly greater than 20% concentrations of blank group at the 1, 3, 5 and 7 days (P<0.05).
20% concentrations of Gubishu containing serum can significantly increase the proliferation of chondrocytes, and bring the logarithmic growth period forward to the 3 day."

Bunch of studies here that I need.

Stk11

Enchondroma resulting from loss of a Stk11-dependent switch of proliferative chondrocytes to a postmitotic fate

"Stk11 (also known as liver kinase b1 (Lkb1)) is a serine-threonine protein kinase that acts upstream of the AMP-activated protein kinase (AMPK) family in coupling energy homeostasis to cell growth, proliferation and survival. Through chondrocyte-specific removal of Lkb1 activity, we showed that Stk11 is required for the normal switch of mitotic chondrocytes to a postmitotic hypertrophic chondrocyte fate. Consequently, it led to a dramatic overgrowth of the growth plate in the Stk11-mutant postnatal skeletal elements. To determine the molecular mechanisms underlying Stk11 action, we examined the mTOR pathway, which is inhibited through AMPK in growth regulation. Strikingly, rapamycin treatment of the pregnant mouse was able to rescue the delay in chondrocyte hypertrophy in Stk11-mutant embryos, suggesting that Stk11 inhibition of mTOR signaling is critical for the switch in chondrocyte fate. Since the dramatic overgrowth of the growth plate is characteristic of enchondroma, we also examined the tumorigenicity of the mutant chondrocytes both in vitro and in vivo. In contrast to wild-type chondrocytes isolated from the postnatal day-30 growth plate, Stk11-mutant chondrocytes proliferated and formed colonies in monolayer and anchorage-independent agar cultures, indicative of a neoplastic transformation in vitro. Similarly, allotransplantation of mutant chondrocytes into immune-deficient NOG mice also resulted in tumor formation in vivo. Gene Ontology analysis of gene expression profiles indicated an augmented activity of cell proliferation and cell cycle regulators within the enchondroma-chondrocyte population compared to chondrocytes in the normal growth plate."

Loss of Stk11 (Lkb1) in Chondrocytes Delays Chondrocyte Hypertrophy Resulting in a Chondrosarcoma-like Overgrowth in the Postnatal Skeleton

"Stk11 is required for the normal switch of mitotic chondrocytes to a post-mitotic hypertrophic chondrocyte fate. We examined the mTOR pathway, which is inhibited by AMPK in growth regulation. Strikingly, rapamycin treatment of the pregnant mouse was able to rescue the delay in chondrocyte hypertrophy in Stk11 mutant embryos, suggesting that Stk11 inhibition of mTOR signaling is critical for the switch in chondrocyte fate. To determine the consequences of Stk11 removal in the postnatal skeleton, we examined long bones following chondrocyte specific removal of Stk11 at embryonic stages. Removal of Stk11 led to chondrosarcoma-like overgrowth phenotype in the Stk11 mutant skeletal elements. Furthermore, in contrast to wild-type chondrocytes isolated from the postnatal day 30 growth plate, Stk11 mutant chondrocytes proliferated and formed colonies in monolayer and anchorage-independent agar cultures, indicative of a neoplastic transformation. Gene Ontology analysis of gene expression profiles indicated an augmented activity of cell proliferation and cell cycle regulators within the chondrosarcoma-like population compared to chondrocytes in the normal growth plate. Stk11 [balances] proliferative and non-proliferative hypertrophic states of chondrocyte development through the regulation of mTOR signaling."

Sunday, March 1, 2009

Thyroid Hormone

 Lithium can inhibit thyroid hormone.

Thyroid hormones regulate fibroblast growth factor receptor signaling during chondrogenesis.

"Childhood hypothyroidism causes growth arrest with delayed ossification and growth-plate dysgenesis, whereas thyrotoxicosis accelerates ossification and growth. Thyroid hormone (T(3)) regulates chondrocyte proliferation and is essential for hypertrophic differentiation. Fibroblast growth factors (FGFs) are also important regulators of chondrocyte proliferation and differentiation, and activating mutations of FGF receptor-3 (FGFR3) cause achondroplasia. T(3) regulates chondrogenesis via FGFR3 in ATDC5 cells, . ATDC5 cells expressed two FGFR1, four FGFR2, and one FGFR3 mRNA splice variants throughout chondrogenesis, and expression of each isoform was stimulated by T(3) during the first 6-12 d of culture, when T(3) inhibited proliferation by 50%. FGFR3 expression was also increased in cells treated with T(3) for 21 d, when T(3) induced an earlier onset of hypertrophic differentiation and collagen X expression. FGFR3 expression was reduced in growth plates from T(3) receptor alpha-null mice, which exhibit skeletal hypothyroidism, but was increased in T(3) receptor beta(PV/PV) mice, which display skeletal thyrotoxicosis. FGFR3 is a T(3)-target gene in chondrocytes.  T(3) enhanced FGF2 and FGF18 activation of the MAPK-signaling pathway but inhibited their activation of signal transducer and activator of transcription-1. FGF9 did not activate MAPK or signal transducer and activator of transcription-1 pathways in the absence or presence of T(3). Thus, T(3) exerted differing effects on FGFR activation during chondrogenesis depending on which FGF ligand stimulated the FGFR and which downstream signaling pathway was activated."

" T3 stimulates chondrogenesis in cultured ATDC5 cells by inhibiting cell proliferation and stimulating the onset of hypertrophic chondrocyte differentiation. The antiproliferative effects of T3 occurred during the first 6 d of ATDC5 cell culture, and differentiation progressed until the onset of terminal hypertrophic chondrocyte differentiation by 21 d in T3-treated cells. T3 stimulation of FGFR1 and FGFR2 in ATDC5 cells undergoing chondrogenesis occurred until d 12, but stimulation of FGFR3 by T3 was greater and persisted until d 21, coinciding with the period in which T3 inhibited chondrocyte proliferation and advanced the onset of hypertrophic differentiation. "

"thyrotoxicosis[overactivity of the thyroid gland] leads to accelerated growth and advanced bone age but causes short stature because of premature growth-plate fusion"

Evaluation of the interaction between thyroid hormone and the sympathetic nervous system, via alpha 2 adrenoceptors, on the regulation of bone growth and maturation

"Bone remodeling is under control of the central nervous system (SNC), with the sympathetic nervous system (SNS) acting as the peripheral effector.  The SNS negatively regulates bone mass, acting exclusively via beta 2-adrenoceptor (B2-AR), which is expressed in osteoblasts. Mice with double gene inactivation of the adrenoceptor alpha2A and alpha2C (a2A /a2C -AR-/-) present a phenotype of high bone mass (HBM), in spite of presenting chronic sympathetic hyperactivity and intact B2-AR. These knockout (KO) mice are resistant to the thyroid hormone (TH)-induced osteopenia. Mice with single inactivation of a2A-AR or a2C-AR are resistant to the lower longitudinal bone growth induced by thyrotoxicosis[overactivation of the thyroid gland]. By immunohistochemistry, we detect that both a2A-AR and a2C-AR are expressed in the bone tissue, in the chondrocytes of the reserve and hypertrophic zones of the epiphyseal growth plates (EGP) and in the hypertrophic chondrocytes of the secondary ossification centers of mice. (i) B2-AR is not the sole adrenoceptor involved in the control of bone metabolism and that (ii) the SNS interacts with TH to regulate not only the bone mass, but also the longitudinal bone growth. (i) a2A-AR and/or a2C-AR present an important role in mediating the actions of the SNS in the skeleton and that (ii) these receptors are involved in the TH-SNS interaction to regulate bone metabolism, growth and development. In the present project, we aim to (i) evaluate if the isolated inactivation of a2A-AR and a2C-AR and if the double inactivation of these receptors interfere in the longitudinal bone growth and in the endochondral and intramembranous ossificafication; (ii) characterize the phenotype of the EGP of a2A-AR-/-, a2C-AR-/- and a2A /a2C -AR-/- mice; (iii) evaluate if the action of TH on bone longitudinal growth depends on a2A-AR and/or a2C-AR, analyzing the effect of TH on the EGP structure and on bone growth of a2A-AR-/-, a2C-AR-/- and a2A /a2C -AR-/- mice; (iv) analyze if known pathways of TH action in the EGP (GH/IGF-1 and Wnt/Beta-catenine pathways) are affected by the isolated inactivation of a2A-AR or a2C-AR or by the double inactivation of these receptors (a2A/C-AR-/-); (v) evaluate if the TH action in the endochondral and intramembranous ossification depends on a2A-AR and/or a2C-AR. "