Cyclooxygenase-2 gene disruption promotes proliferation of murine calvarial osteoblasts in vitro

Cyclooxygenase-2 (COX-2) is highly expressed in osteoblasts, and COX-2 produced prostaglandins (PGs) can increase osteoblastic differentiation in vitro. The goal of this study was to examine effects of COX-2 expression on calvarial osteoblastic proliferation and apoptosis. Primary osteoblasts (POBs) were cultured from calvariae of COX-2 wild-type (WT) and knockout (KO) mice. POB proliferation was evaluated by (3)H-thymidine incorporation and analysis of cell replication and cell cycle distribution by flow cytometry. POB apoptosis was evaluated by annexin and PI staining on flow cytometry. As expected, PGE(2) production and alkaline phosphatase (ALP) activity were increased in WT cultures compared to KO cultures. In contrast, cell numbers were decreased in WT compared to KO cells by day 4 of culture. Proliferation, measured on days 3-7 of culture, was 2-fold greater in KO than in WT POBs and associated with decreased Go/G1 and increased S cell cycle distribution. There was no significant effect of COX-2 genotype on apoptosis under basal culture conditions on day 5 of culture. Cell growth was decreased in KO POBs by the addition of PGE(2) or a protein kinase A agonist and increased in WT POBs by the addition of NS398, a selective COX-2 inhibitor. In contrast, differentiation and cell growth in marrow stromal cell (MSC) cultures, evaluated by ALP and crystal violet staining respectively, were increased in MSCs from WT mice compared to MSCs from KO mice, and exogenous PGE(2) increased cell growth in KO MSC cultures. We conclude that PGs secondary to COX-2 expression decrease osteoblastic proliferation in cultured calvarial cells but increase growth of osteoblastic precursors in MSC cultures.     

Effects of high-frequency near-infrared diode laser irradiation on the proliferation and migration of mouse calvarial osteoblasts

Laser irradiation activates a range of cellular processes and can promote tissue repair. Here, we examined the effects of high-frequency near-infrared (NIR) diode laser irradiation on the proliferation and migration of mouse calvarial osteoblastic cells (MC3T3-E1). MC3T3-E1 cells were cultured and exposed to high-frequency (30 kHz) 910-nm diode laser irradiation at a dose of 0, 1.42, 2.85, 5.7, or 17.1 J/cm². Cell proliferation was evaluated with BrdU and ATP concentration assays. Cell migration was analyzed by quantitative assessment of wound healing using the Incucyt^® ZOOM system. In addition, phosphorylation of mitogen-activated protein kinase (MAPK) family members including p38 mitogen-activated protein kinase (p38), stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK), and extracellular signal-regulated protein kinase (ERK)1/2) after laser irradiation was examined with western blotting. Compared to the control, cell proliferation was significantly increased by laser irradiation at a dose of 2.85, 5.7, or 17.1 J/cm². Laser irradiation at a dose of 2.85 J/cm² induced MC3T3-E1 cells to migrate more rapidly than non-irradiated control cells. Irradiation with the high-frequency 910-nm diode laser at a dose of 2.85 J/cm² induced phosphorylation of MAPK/ERK1/2 15 and 30 min later. However, phosphorylation of p38 MAPK and SAPK/JNK was not changed by NIR diode laser irradiation at a dose of 2.85 J/cm². Irradiation with a high-frequency NIR diode laser increased cell division and migration of MT3T3-E1 cells, possibly via MAPK/ERK signaling. These observations may be important for enhancing proliferation and migration of osteoblasts to improve regeneration of bone tissues.     

Molybdenum-induced changes in the epiphyseal growth plate

Summary Molybdenum (Mo), at high concentrations, induces changes in the epiphyseal growth plate through its effects on copper (Cu) metabolism but it is unclear whether or not Mo can induce changes independent of its effects on copper status. To this end, the effect of Mo on longitudinal bone growth was examined in rats. Dietary Mo was given either as ammonium heptamolybdate or as ammonium tetrathiomolybdate, the latter producing a marked Cu deficiency. There was a significant reduction in longitudinal bone growth in both groups; however, growth plate width was increased only in the Cu-deficient animals due to an increase in the width of the zone of transitional/hypertrophic chondrocytes. Both glucose 6-phosphate dehydrogenase activity and cell proliferation (assessed by bromodeoxyuridine incorporation) were markedly decreased in the proliferating zone of the growth plate in both Mo-treated groups. These changes were not apparently related to changes in circulating vitamin D metabolites or insulin-like growth factor-1. The results indicate that excess Mo impairs cell proliferation within the growth plate, whereas the effects of copper deficiency are more related to chondrocyte differentiation. Thus, Mo can induce changes in longitudinal bone growth which are distinct from those resulting from Cu deficiency.     

淫羊藿苷含药血清对体外培养大鼠颅骨成骨细胞增殖与分化的影响

目的 研究淫羊藿苷含药血清(Serum of rats administered icariin,SI)对体外培养大鼠颅骨成骨细胞(Rat calvarial osteoblasts,ROB)增殖和分化成熟的影响.方法 以每1 kg体重0.25 g淫羊藿苷的剂量灌服成年大鼠制得淫羊藿苷含药血清,以灌服等体积生理盐水制得对照血清.分别以2.5%,5%和10%3种浓度加入ROB培养基,检测对成骨细胞增殖、碱性磷酸酶(ALP)活性和钙化结节数等的影响.结果 2.5%和5%SI均促进细胞的增殖,尤以5%浓度更为明显,该浓度含药血清提高ROB的ALP活性,增加Ⅰ型胶原表达,并显著提高钙化结节形成数量.结论 淫羊藿经口服后的代谢产物可刺激成骨细胞增殖,促进其分化成熟,是淫羊藿抗骨质疏松的有效成分.     

Fidgetin-like 1 gene inhibited by basic fibroblast growth factor regulates the proliferation and differentiation of osteoblasts.

The FIGNL1 gene was proven to be a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). In this in vitro study, the AAA proteins inhibited osteoblast proliferation and stimulated osteoblast differentiation. We showed that FIGNL1 may play some regulatory role in osteoblastogenesis. INTRODUCTION: The fidgetin-like 1 (FIGNL1) gene encodes a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). Although the FIGNL1 protein localizes to both the nucleus and cytoplasm, the function of FIGNL1 remains unknown. In a previous study, we identified several genes that mediate the anabolic effects of basic fibroblast growth factor (bFGF) on bone by using microarray data. FIGNL1 was one of the genes that downregulated >2-fold in MC3T3-E1 cells after treatment with bFGF. Therefore, this study was aimed to identify and confirm the function of FIGNL1 on osteoblastogenesis. MATERIALS AND METHODS: We examined the effect of the FIGNL1 gene on proliferation, differentiation, and apoptosis in mouse osteoblast cells (MC3T3-E1 and mouse primary calvarial cells) using flow cytometry, RT-PCR, cell proliferation assay, and cell death assay. MC3T3-E1 cells and mouse calvarial cells were transfected with small interfering RNA (siRNA) directed against the FIGNL1 or nontargeting control siRNA and examined by cell proliferation and cell death assays. Also, FIGNL1 was fused to enhance green fluorescent protein (EGFP), and the EGFP-fused protein was transiently expressed in MC3T3-E1 cells. RESULTS: Reduced expression of FIGNL1 by bFGF and TGF-beta1 treatment was verified by RT-PCR analysis. Overexpression of FIGNL1 reduced the proliferation of MC3T3-E1 and calvarial cells, more than the mock transfected control cells did. In contrast, siFIGNL1 transfection significantly increased the proliferation of osteoblasts, whereas overexpression of FIGNL1 did not seem to alter apoptosis in osteoblasts. Meanwhile, overexpression of FIGNL1 enhanced the mRNA expression of alkaline phosphatase (ALP) and osteocalcin (OCN) in osteoblasts. In contrast, siFIGNL1 decreased the expression of ALP and OCN. A pEGFP-FIGNL1 transfected into MCT3-E1 cells had an initially ubiquitous distribution and rapidly translocated to the nucleus 1 h after bFGF treatment. CONCLUSIONS: From these results, we proposed that FIGNL1, a subfamily member of the AAA family of proteins, might play some regulatory role in osteoblast proliferation and differentiation. Further analyses of FIGNL1 will be needed to better delineate the mechanisms contributing to the inhibition of proliferation and stimulation of osteoblast differentiation.     

C反应蛋白激活原纤毛抑制颅骨成骨细胞的增殖和分化

目的 研究C反应蛋白(C-reactive protein,CRP)对原代培养SD大鼠乳鼠颅骨成骨细胞(rat calvarial osteoblasts,ROB)增殖及成骨分化的影响.方法 取SD乳鼠颅骨组织用胰蛋白酶和胶原蛋白酶Ⅰ消化得到原代ROB,随后将ROB用α-MEM培养基培养,传代3次后用于后续实验.在对照...     

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 ～ 2–6 fold, and reduced to ～ 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. 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 ～ 55–75%, and also ～ 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. Taken together these data suggest that Mustn1 is a necessary regulator of chondrocyte function.     

Production of both interleukin-1 alpha and beta by newborn mouse calvarial cultures

The conditioned medium (CM) from 4-6 day newborn mouse calvarial cultures was found to contain thymocyte comitogen proliferation activity. This activity was blocked by an antiserum to murine interleukin-1 alpha (IL-1 alpha) but not by an antiserum to murine interleukin-1 beta. The release of thymocyte comitogen proliferation activity from the cultures did not appear dependent on endotoxin and was not associated with detectable interleukin-2 activity in the CM. Activity in the CM eluted from a gel filtration column with a peak Mr of 16-18 kD (the Mr of mature murine IL-1 alpha and beta is 17 kD). Western immunoblots of 100-fold concentrated CM demonstrated only a single 33 kD band with an antiserum to murine IL-1 beta and no bands with an antiserum to murine IL-1 alpha. However, this assay was relatively insensitive (limit of detection 1-10 ng compared with 1-10 pg for the thymocyte comitogen proliferation assay). Immunoprecipitation of [35S]methionine-labeled CM with three different anti-IL-1 alpha antisera, a more sensitive assay, demonstrated 15-17 kD bands in all cases. These results demonstrate that 4-6 day newborn mouse calvarial cultures spontaneously release 17 kD IL-1 alpha and 33 kD IL-1 beta into their conditioned medium. It appears that although 17 kD IL-1 alpha is the major bioactive form in the CM, 33 kD IL-1 beta is present in greater amounts. These results also suggest that local production of IL-1 can regulate bone cell function and may play a role in bone growth and remodeling.     

The proto-oncogene C-raf-1 is highly expressed only in the hypertrophic zone of the growth plate

Proto-oncogene c-raf-1, the cellular homologue of the acutely transforming oncogene v-raf, has a central role in the signal tranduction pathways. The growth plate, due to its non-overlapping zones of chondrocyte maturation, provides a physiological in situ model for investigating the role of c-raf-1 in proliferation and differentiation of chondrocytes. In this study, Northern blotting was first performed to examine the expression of mRNA for c-raf-1 in the embryonic chick tibial growth plate. It revealed that the normal levels of c-raf-1 mRNA were associated with the whole growth plate. We then investigated the localization of c-raf-1 mRNA and c-raf-1 protein in the growth plate by in situ hybridization and immunohistochemistry in order to determine whether c-raf-1 is involved in chondrocyte maturation. Our results showed that c-raf-1 mRNA and c-raf-1 protein were detected only in the hypertrophic zone. The data suggest involvement of this proto-oncogene in chondrocyte differentiation and/or hypertrophy rather than in proliferation.     

Expression of estrogen receptor alpha and beta in the epiphyseal plate of the rat

In this study we examine the spatial and temporal expression of estrogen receptor (ER) alpha and beta mRNA and protein in the tibial growth plate of the rat after birth, as well as the hormonal regulation of their expression. Using in situ hybridization and immunohistochemistry, we demonstrated ER alpha and ER beta mRNA and protein in tibial growth plates from 1 to 40 weeks after birth. ER alpha and beta mRNA and protein were localized in late proliferating and early hypertrophic chondrocytes during early life (1 and 4 weeks of age), whereas the immunohistochemistry also showed staining for ER alpha and beta in the resting cells. A similar expression pattern was observed during sexual maturation (7 weeks of age) except that ER beta mRNA was also detected in early proliferating chondrocytes. After sexual maturation (from 12 up to 40 weeks of age) ER alpha and beta mRNA and protein expression was confined to late proliferating and early hypertrophic chondrocytes. Apart from a relatively higher ER alpha mRNA expression in males after sexual maturation, we did not detect differences in expression of ERs between genders. Expression of ER beta mRNA in epiphyseal plates was increased in growth-retarded hypophysectomized rats compared with controls. Administration of growth hormone (GH) did not reverse the increased ER expression to normal. These data suggest that ER alpha and beta are coexpressed in growth plates of the rat after birth and that the level of expression of ERs in these tissues is hormonally regulated. Furthermore, our data indicate that the absence of growth-plate closure in the rat cannot be explained by disappearance of ER alpha expression during sexual maturation per se.     

Impact of growth plate senescence on catch-up growth and epiphyseal fusion

In mammals, longitudinal bone growth occurs rapidly in prenatal and early postnatal life, but then slows and eventually ceases. This deceleration, which reflects a decline in chondrocyte proliferation, was previously attributed to a hormonal or other systemic mechanism. However, new evidence suggests that it is due to a local mechanism within the growth plate. In particular, recent findings suggest that growth plate chondrocytes have a finite proliferative capacity that is gradually exhausted, causing growth to slow and finally stop. This concept has provided insight into clinical phenomena including catch-up growth after transient growth inhibition, catch-down growth after transient estrogen exposure, and epiphyseal fusion.J. Baron is a commissioned officer in the U.S. Public Health ServiceThis work was presented in part at the IPNA Seventh Symposium on Growth and Development in Children with Chronic Kidney Disease: The Molecular Basis of Skeletal Growth, 1–3 April 2004, Heidelberg, Germany     

Comparison of the effects of excess vitamin A and high oxygen tension onin vitro epiphyseal plate growth

Epihyseal plate organ cultures grown in medium containing excess vitamin A (100 USP units/ml) showed marked reduction in the length of the cartilage component of the explant, progressive loss of acid mucopolysaccharide stainability of the cartilage matrix, loss of the zone of hypertrophic cells followed by the loss of all zoning, an accumulation of cells at the bone-cartilage junction, and eventual detachment of the cartilage from the bone. Epsilon partially reversed these effects. Explants grown in 90% oxygen showed no reduction in the length of the cartilage component, a progressive loss of acid mucopolysaccharide stainability of the cartilage matrix narrowing and eventual loss of the zone of hypertrophic cells, an accumulation of an acellular, PAS-positive debris at the bone-cartilage junction, and marked reduction in length of the bone component of the explant. EACA and chloroquine partially reversed these effects. The role of lysosomal enzymes in mediating these effects is discussed. It is suggested that different lysosomal enzymes may be operational in the presence of excess vitamin A than in the presence of high oxygen tension.     

PTH/PTHrP receptor mRNA is down-regulated in epiphyseal cartilage growth plate of uraemic rats

PTH/PTHrP receptor mRNA is down-regulated in epiphyseal cartilagegrowth plate of uraemic rats. Growth retardation, hypocalcaemia,hyperphosphataemia, and skeletal resistance to the action ofPTH are well known features of advanced chronic renal failure(CRF). It has been suggested that the downregulation of renaland skeletal PTH receptors (PTH/PTHrP-R) could play an importantrole in the occurrence of these abnormalities. In the presentstudy, four uraemic (4 weeks after 5/6 nephrectomy) and fourcontrol (sham-operated) rats were analysed for PTH/PTHrP-R mRNAexpression at the proximal femoral and tibial growth platesby in situ hybridization. Uraemic rats had plasma biochemicalabnormalities of advanced CRF including high creatinine, phosphate,and PTH, and low calcium and calcitriol levels. The femoraland tibial bones of uraemic animals were shorter in length thanthose of control rats, and had reduced width and cellularityof the epiphyseal cartilage growth plate. Mean (±SD)tibia growth plate width was 152±30 µm in uraemicrats, compared with 170±35 µm in control rats.The difference was mostly due to a marked reduction of the zoneexpressing PTH/PTHrP-R (mature chondrocytes) which was 30±5µm in tibias from uraemic versus 44±10 µmin tibias from control rats. The hybridization signals of PTH/PTHrP-Rper individual cell were quantified on dark field images usinga computer-assisted image analysis system. The number of grainsin PTH/PTHrP-R positive cells was also decreased in uraemicrats, 103±13 compared with 123±14 arbitrary units(dark pixel density)/cell in control rats (P 0.005). In conclusion,these data indicate that rats with severe CRF and secondaryhyperparathyroidism have reduced epiphyseal cartil age PTH/PTHrP-RmRNA expression. This alteration may be relevant in the pathogenesisof growth retardation in uraemia.     

Heparin facilities proliferation of human osteoblasts in vitro

It is a well-known fact that long-term application of heparin can lead to osteoporosis. To learn more about the mechanisms of heparin-induced osteoporosis, we exposed human osteoblasts in vitro to heparin in various concentrations. We found an increased proliferation rate, especially in concentrations used therapeutically in humans (0.1-0.2 IU/ml). In our experiments fetal calf serum (FCS) was able to heighten the positive effect of heparin, showing a synergism between heparin and FCS.