Stimulatory effects of insulin-like growth factor-I on growth plate chondrogenesis are mediated by nuclear factor-kappaB p65

Insulin-like growth factor-I (IGF-I) is an important regulator of endochondral ossification. However, little is known about the signaling pathways activated by IGF-I in growth plate chondrocytes. We have previously shown that NF-kappaB-p65 facilitates growth plate chondrogenesis. In this study, we first cultured rat metatarsal bones with IGF-I and/or pyrrolidine dithiocarbamate (PDTC), a known NF-kappaB inhibitor. The IGF-I-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by PDTC. In rat growth plate chondrocytes, IGF-I induced NF-kappaB-p65 nuclear translocation. The inhibition of NF-kappaB-p65 expression and activity (by p65 short interfering RNA and PDTC, respectively) in chondrocytes reversed the IGF-I-mediated induction of cell proliferation and differentiation and the IGF-I-mediated prevention of cell apoptosis. Moreover, the inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of IGF-I on NF-kappaB activation. In conclusion, our findings indicate that IGF-I stimulates growth plate chondrogenesis by activating NF-kappaB-p65 in chondrocytes.     

Igf1 promotes longitudinal bone growth by insulin-like actions augmenting chondrocyte hypertrophy.

Longitudinal bone growth, and hence stature, are functions of growth plate chondrocyte proliferation and hypertrophy. Insulin-like growth factor 1 (Igf1) is reputed to augment longitudinal bone growth by stimulating growth plate chondrocyte proliferation. In this study, however, we demonstrate that chondrocyte numbers and proliferation are normal in Igf1 null mice despite a 35% reduction in the rate of long bone growth. Igf1 null hypertrophic chondrocytes differentiate normally in terms of expressing specialized proteins such as collagen X and alkaline phosphatase, but are smaller than wild-type at all levels of the hypertrophic zone. The terminal hypertrophic chondrocytes, which form the scaffold on which long bone growth extends, are reduced in linear dimension by 30% in Igf1 null mice, accounting for most of their decreased longitudinal growth. The expression of the insulin-sensitive glucose transporter, GLUT4, is significantly decreased and the insulin-regulated enzyme glycogen synthase kinase 3beta (GSK3) is hypo-phosphorylated in Igf1 null chondrocytes. Glycogen levels were significantly decreased and ribosomal RNA levels were reduced by almost 75% in Igf1 null chondrocytes. These data suggest that Igf1 promotes longitudinal bone growth by 'insulin-like' anabolic actions which augment chondrocyte hypertrophy.     

Annexin-mediated Ca2+ influx regulates growth plate chondrocyte maturation and apoptosis

Maturation of epiphyseal growth plate chondrocytes plays an important role in endochondral bone formation. Previously, we demonstrated that retinoic acid (RA) treatment stimulated annexin-mediated Ca(2+) influx into growth plate chondrocytes leading to a significant increase in cytosolic Ca(2+), whereas K-201, a specific annexin Ca(2+) channel blocker, inhibited this increase markedly. The present study addressed the hypothesis that annexin-mediated Ca(2+) influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. We found that K-201 significantly reduced up-regulation of expression of terminal differentiation marker genes, such as cbfa1, alkaline phosphatase (APase), osteocalcin, and type I collagen in RA-treated cultures. Furthermore, K-201 inhibited up-regulation of annexin II, V, and VI gene expression in these cells. RA-treated chondrocytes released mineralization-competent matrix vesicles, which contained significantly higher amounts of annexins II, V, and VI as well as APase activity than vesicles isolated from untreated or RA/K-201-treated cultures. Consistently, only RA-treated cultures showed significant mineralization. RA treatment stimulated the whole sequence of terminal differentiation events, including apoptosis as the final event. After a 6-day treatment gene expression of bcl-2, an anti-apoptotic protein, was down-regulated, whereas caspase-3 activity and the percentage of TUNEL-positive cells were significantly increased in RA-treated cultures compared with untreated cultures. Interestingly, the cytosolic calcium chelator BAPTA-AM and K-201 protected RA-treated chondrocytes from undergoing apoptotic changes, as indicated by higher bcl-2 gene expression, reduced caspase-3 activity, and the percentage of TUNEL-positive cells. In conclusion, annexin-mediated Ca(2+) influx into growth plate chondrocytes is a positive regulator of terminal differentiation, mineralization, and apoptosis events in growth plate chondrocytes.     

Obovatol inhibits colorectal cancer growth by inhibiting tumor cell proliferation and inducing apoptosis

Neolignans such as obovatol, honokiol, and magnolol have been previously reported to show various biological activities including anti-inflammation and antitumor effects. This is the first demonstration on the in vivo antitumor effect of obovatol on human colorectal carcinoma SW620 cells. Nude mice were implanted with SW620 cells and fed with vehicle or 5mg/kg/d dose of obovatol for 20 days. Obovatol inhibited tumor growth that accounted for 50% decrease in tumor volume and 44.6% decrease in tumor weight at the end of the experiment without any adverse health effect. In nude mice bearing SW620-incubated tumor, obovatol exhibited more potent antitumor activity than honolkiol. In addition, DNA flow cytometric analysis shows that obovatol progresses to apoptosis as detected by flow cytometry after double staining with annexin V and propidium iodide. Thus, we suggest that obovatol is a potent inducer of cell apoptosis in SW620 cells, and a potent antitumor agent.     

Runx3 regulates chondrocyte phenotype by controlling multiple genes involved in chondrocyte proliferation and differentiation

Molecular Biology Reports - Chondrocytes are the sole cell type present within cartilage, and play pivotal roles in controlling the formation and composition of health cartilage. Chondrocytes...     

Proteomic analysis of the effects of antler extract on chondrocyte proliferation,differentiation and apoptosis

Molecular Biology Reports - Deer antlers are unique cranial appendages capable of regeneration and rapid growth. In addition, deer antlers have been widely used in traditional Chinese medicine to...     

Nuclear co-translocation of myotrophin and p65 stimulates myocyte growth. Regulation by myotrophin hairpin loops

Myotrophin, a 12-kDa ankyrin repeat protein, stimulates protein synthesis and cardiomyocyte growth to initiate cardiac hypertrophy by activating the NF-kappaB signaling cascade. We found that, after internalization into myocytes, myotrophin cotranslocates into the nucleus with p65 to stimulate myocyte growth. We used structure-based mutations on the hairpin loops of myotrophin to determine the effect of the loops on myotrophin and p65 localization, induction of protein synthesis, and cardiac hypertrophy. Loop mutants, most prominently glutamic acid 33-->alanine (E33A), stimulated protein synthesis much less than wild type. Myotrophin-E33A internalized into myocytes but did not translocate into the nucleus and failed to promote nuclear translocation of p65. In addition, two cardiac hypertrophy marker genes, atrial natriuretic factor and beta-myosin heavy chain, were not up-regulated in E33A-treated cells. Myotrophin-induced myocyte growth and initiation of hypertrophy thus require nuclear co-translocation of myotrophin and p65, in a manner that depends crucially on the myotrophin hairpin loops.     

Role of cholesterol in the regulation of growth plate chondrogenesis and longitudinal bone growth

Inborn errors of cholesterol synthesis are associated with multiple systemic abnormalities, including skeletal malformations. The regulatory role of cholesterol during embryogenesis appears to be mediated by Shh, a signaling molecule in which activity depends on molecular events involving cholesterol. Based on this evidence, we hypothesized that cholesterol, by modifying the activity of Ihh (another of the Hedgehog family proteins) in the growth plate, regulates longitudinal bone growth. To test this hypothesis, we treated rats with AY 9944, an inhibitor of the final reaction of cholesterol synthesis. After 3 weeks, AY 9944 reduced the cumulative growth, tibial growth, and the tibial growth plate height of the rats. To determine whether cholesterol deficiency affects bone growth directly at the growth plate, we then cultured fetal rat metatarsal bones in the presence of AY 9944. After 4 days, AY 9944 suppressed metatarsal growth and growth plate chondrocyte proliferation and hypertrophy. The inhibitory effect on chondrocyte hypertrophy was confirmed by the AY 9944-mediated decreased expression of collagen X. Lastly, AY 9944 decreased the expression of Ihh in the metatarsal growth plate. We conclude that reduced cholesterol synthesis in the growth plate, possibly by altering the normal activity of Ihh, results in suppressed longitudinal bone growth and growth plate chondrogenesis.     

Herba epimedii flavonoids suppress osteoclastic differentiation and bone resorption by inducing G2/M arrest and apoptosis

Accumulating evidences suggest that Herba epimedii has the potential benefits against osteoporosis. However, previous studies were focused on the crude extract, total flavonoids (TF) and icariin (ICA), and the detailed molecular mechanisms of action and structure–activity relationship (SAR) remain unclear. Herein we aimed to systematically investigate the effects of Herba epimedii flavonoids (HEF) on the activity of osteoclasts, and explore the potential SAR. Both ICA and baohuoside-1 (BS) significantly inhibited the proliferation of RAW 264.7 cells (IC₅₀ 25 μM and 67 μM, respectively). Treatment of ICA resulted in G2/M arrest and apoptosis in RAW 264.7 cells as early as 12 h. Besides, HEF remarkably suppressed vitamin D-induced differentiation of osteoclasts in rabbit bone marrow cells and the bone resorption of rabbit mature osteoclasts in vitro. It is notable that the inhibitory effect of 100 μM ICA and BS on osteoclast formation is almost 90%; and the inhibition rate on bone resorption is 50% and 80%, respectively. Besides, RANKL-induced osteoclast formation from RAW 264.7 cells and the expression of TRAP, CA II, CTSK and MMP-9 was significantly reduced by the treatment of 25 μM HEF and 17β-estradiol (ES), and the inhibitory strength increases in the order TF < ES < ICA < BS, which was blocked by ICI182780 suggesting that the regulation of osteoclast activity might be ER dependent. Furthermore, the free hydroxyl group at C-7 of BS played an important role in the SAR for anti-osteoclast action. To conclude, HEF could regulate the formation and activity of osteoclasts by inhibiting the proliferation and differentiation, inducing apoptosis and cell cycle arrest and suppressing bone resorption of osteoclasts. Changes in osteoclast activity are probably mediated predominantly by interaction with nuclear estrogen receptors and via mitochondrial pathway. HEF, especially BS, has great potential for the prevention and treatment of osteoporosis.     

MiR‐29b‐3p promotes chondrocyte apoptosis and facilitates the occurrence and development of osteoarthritis by targeting PGRN

This study was aimed to explore the role of miR‐29b‐3p and PGRN in chondrocyte apoptosis and the initiation and progress of osteoarthritis (OA). Both miR‐29b‐3p and PGRN were up‐regulated in cartilage tissue from patients with OA. Transfection of miR‐29b‐3p mimic into rat primary chondrocytes and SW1353 chondrosarcoma cells significantly suppressed PGRN expression and release, induced apoptosis, inhibited proliferation and scratch wound closure. By contrast, transfection of miR‐29b‐3p inhibitor exhibited the opposite effects. Moreover, the expression and secretion of cartilaginous degeneration‐related molecules were also altered by miR‐29b‐3p. Luciferase reporter gene assay showed rat GRN mRNA is directly targeted and repressed by miR‐29b‐3p. The fact that recombinant PGRN or shPGRN‐mediated PGRN interference abolished miR‐29b‐3p mimic‐induced cell apoptosis and growth inhibition suggested miR‐29b‐3p affect the cellular functions of chondrocyte through regulating PGRN expression. In vivo, joint cavity injection of miR‐29b‐3p antagomir prior to surgical induction of OA significantly suppressed the upregulation of miR‐29b‐3p, whereas further promoted the increased expression of PGRN. Articular chondrocytes apoptosis and cartilage loss in the knee joint of surgically induced OA rats were also ameliorated by the injection of miR‐29b‐3p antagomir, demonstrated by TUNEL and safranin O‐fast green staining. This work showed miR‐29b‐3p facilitates chondrocyte apoptosis and OA by targeting PGRN, and miR‐29b‐3p or PGRN may be the potential target for OA treatments.     

FGF18 is required for early chondrocyte proliferation, hypertrophy and vascular invasion of the growth plate

Fibroblast growth factor 18 (FGF18) has been shown to regulate chondrocyte proliferation and differentiation by signaling through FGF receptor 3 (FGFR3) and to regulate osteogenesis by signaling through other FGFRs. Fgf18(-/-) mice have an apparent delay in skeletal mineralization that is not seen in Fgfr3(-/-) mice. However, this delay in mineralization could not be simply explained by FGF18 signaling to osteoblasts. Here we show that delayed mineralization in Fgf18(-/-) mice was closely associated with delayed initiation of chondrocyte hypertrophy, decreased proliferation at early stages of chondrogenesis, delayed skeletal vascularization and delayed osteoclast and osteoblast recruitment to the growth plate. We further show that FGF18 is necessary for Vegf expression in hypertrophic chondrocytes and the perichondrium and is sufficient to induce Vegf expression in skeletal explants. These findings support a model in which FGF18 regulates skeletal vascularization and subsequent recruitment of osteoblasts/osteoclasts through regulation of early stages of chondrogenesis and VEGF expression. FGF18 thus coordinates neovascularization of the growth plate with chondrocyte and osteoblast growth and differentiation.     

Fate of the hypertrophic chondrocyte: microenvironmental perspectives on apoptosis and survival in the epiphyseal growth plate

The goal of this review is to examine the fate of the hypertrophic chondrocyte in the epiphyseal growth plate and consider the impact of the cartilage microenvironment on cell survival and apoptosis. Early investigations pointed to a direct role of the hypertrophic chondrocyte in osteogenesis. The terminally differentiated cells were considered to undergo a dramatic change in shape, size, and phenotype, and assume the characteristics of an osteoblast. While some studies have supported the notion of transdifferentiation, much of the evidence in favor of reprogramming epiphyseal chondrocytes is circumstantial and based on microscopic evaluation of cells that are present at the chondro-osseous junction. Although these investigations provided a novel perspective on endochondral bone formation, they were flawed by the failure to consider the importance of stem cells in osseous tissue formation. Subsequent studies indicated that many, if not all, of the cells of the cartilage plate die through the induction of apoptosis. With respect to agents that mediate apoptosis, at the chondro-osseous junction, solubilization of mineral and hydrolysis of organic matrix constituents by septoclasts generates high local concentrations of ions, peptides, and glycans, and secreted matrix metalloproteins. Individually, and in combination, a number of these agents serve as potent chondrocyte apoptogens. We present a new concept: hypertrophic cells die through the induction of autophagy. In the cartilage microenvironment, combinations of local factors cause chondrocytes to express an initial survival phenotype and oxidize their own structural macromolecules to generate ATP. While delaying death, autophagy leads to a state in which cells are further sensitized to changes in the local microenvironment. One such change is similar to ischemia reperfusion injury, a condition that leads to tissue damage and cell death. In the growth cartilage, an immediate effect of this type of injury is sensitization to local apoptogens. These two concepts (type II programmed cell death and ischemia reperfusion injury) emphasize the importance of the local microenvironment, in particular pO(2), in directing chondrocyte survival and apoptosis.     

Arginine deiminase inhibits cell proliferation by arresting cell cycle and inducing apoptosis.

We have previously demonstrated that arginine deiminase inhibits the proliferation of vascular endothelial cells, but the mechanisms leading to growth inhibition have remained unclear. We report here that low concentrations of arginine deiminase purified from Mycoplasma arginini inhibit proliferation of various cultured cells by arresting the cell cycle in G(1) and/or S phase with higher arginine deiminase concentrations leading to subsequent apoptosis. Our results demonstrate that arginine deiminase inhibits cell proliferation not only by depletion of arginine, but also by mechanisms involving the cell cycle and death signals.     

Isocostunolide inhibited glioma stem cell by suppression proliferation and inducing caspase dependent apoptosis

Glioblastoma multiform (GBM) is a highly aggressive brain tumor with poor life expectancy, and glioma stem cells (GSCs) are a small population of tumor cells existed in GBM, in which GSCs response to drive GBM recurrence, invasion and contribute to the anti-cancer resistance. GSCs have been identified and developed as a therapeutic target for GBM and can be used in drugs screening. Isocostunolide is a natural sesquiterpenoid and contained abundant resource in medicinal plants, but the anti-cancer efficacies of it against GSCs are still unexplored. In this investigation, the anti-tumor activity of isocostunolide against GSCs was investigated and the result demonstrated that it inhibited the growth of GSCs (GSC-3#, GSC-12#, GSC-18#) significantly with an IC₅₀ value of 2.80 μg/ml, 2.61 μg/ml, 1.07 μg/ml, respectively. In further mechanism study, isocostunolide inhibited GSCs cell proliferation, induced GSCs apoptosis significantly, as well as increased the proportion of the cleavage of caspase-3. The result suggested that isocostunolide induced GSCs apoptosis via the caspase dependent apoptotic pathway. Moreover, isocostunolide damaged GSCs colony formation capacity significantly and exhibited the anti-cancer efficacy against GSCs in vitro.     

Smad7 Inhibits chondrocyte differentiation at multiple steps during endochondral bone formation and down-regulates p38 MAPK pathways

Bone morphogenetic proteins (BMPs) play critical roles at various stages in endochondral bone formation. In vitro studies have demonstrated that Smad7 regulates transforming growth factor-beta and BMP signals by inhibiting Smad pathways in chondrocytes. However, the in vivo roles of Smad7 during cartilage development are unknown. To investigate distinct effects of Smad7 at different stages during chondrocyte differentiation, we generated a series of conditional transgenic mice that overexpress Smad7 in chondrocytes at various steps of differentiation by using the Cre/loxP system. We generated Col11a2-lacZ(floxed)-Smad7 transgenic mice and mated them with three types of Cre transgenic mice to obtain Smad7(Prx1), Smad7(11Enh), and Smad7(11Prom) conditional transgenic mice. Smad7(Prx1) mice overexpressing Smad7 in condensing mesenchymal cells showed disturbed mesenchymal condensation associated with decreased Sox9 expression, leading to poor cartilage formation. Smad7(11Enh) mice overexpressing Smad7 in round chondrocytes showed decreased chondrocyte proliferation rates. Smad7(11Prom) mice overexpressing Smad7 in flat chondrocytes showed inhibited maturation of chondrocytes toward hypertrophy. Micromass culture of mesenchymal cells showed that BMP-induced cartilaginous nodule formation was down-regulated by overexpression of Smad7, but not Smad6. Overexpression of Smad7, but not Smad6, down-regulated the phosphorylation of p38 MAPKs. Our data provide in vivo evidence for distinct effects of Smad7 at different stages during chondrocyte differentiation and suggest that Smad7 in prechondrogenic cells inhibits chondrocyte differentiation possibly by down-regulating BMP-activated p38 MAPK pathways.     

Nfib promotes chondrocyte proliferation and inhibits differentiation by mildly regulating Sox9 and its downstream genes

Molecular Biology Reports - Chondrocyte proliferation and differentiation play pivotal roles in regulating cartilage formation, endochondral bone formation, and repair. Cartilage damage and...     

生长因子对骨髓间充质干细胞增殖、分化的影响

骨髓间充质干细胞(bone mesenchymal stemcell,BMSC)是骨髓基质细胞的重要组成部分,由于其不但能与其他细胞一起支持造血干细胞造血,而且还具有较强的增殖功能及多向分化潜能,在一定诱导因素下可定向分化成骨细胞、软骨细胞和脂肪细胞等,近年来已成为生物学和医学的研究热点。本文简要介绍了不同生长因子如血管内皮生长因子、碱性成纤维细胞生长因子、转化生长因子-β等对BMSC增殖、分化的影响。