gp130 Cytokines Activate Novel Signaling Pathways and Alter Bone Dissemination in ER+ Breast Cancer Cells

Breast cancer cells frequently home to the bone marrow, where they encounter signals that promote survival and quiescence or stimulate their proliferation. The interleukin-6 (IL-6) cytokines signal through the co-receptor glycoprotein130 (gp130) and are abundantly secreted within the bone microenvironment. Breast cancer cell expression of leukemia inhibitory factor (LIF) receptor (LIFR)/STAT3 signaling promotes tumor dormancy in the bone, but it is unclear which, if any of the cytokines that signal through LIFR, including LIF, oncostatin M (OSM), and ciliary neurotrophic factor (CNTF), promote tumor dormancy and which signaling pathways are induced. We first confirmed that LIF, OSM, and CNTF and their receptor components were expressed across a panel of breast cancer cell lines, although expression was lower in estrogen receptor–negative (ER^–) bone metastatic clones compared with parental cell lines. In estrogen receptor–positive (ER⁺) cells, OSM robustly stimulated phosphorylation of known gp130 signaling targets STAT3, ERK, and AKT, while CNTF activated STAT3 signaling. In ER^– breast cancer cells, OSM alone stimulated AKT and ERK signaling. Overexpression of OSM, but not CNTF, reduced dormancy gene expression and increased ER⁺ breast cancer bone dissemination. Reverse-phase protein array revealed distinct and overlapping pathways stimulated by OSM, LIF, and CNTF with known roles in breast cancer progression and metastasis. In breast cancer patients, downregulation of the cytokines or receptors was associated with reduced relapse-free survival, but OSM was significantly elevated in patients with invasive disease and distant metastasis. Together these data indicate that the gp130 cytokines induce multiple signaling cascades in breast cancer cells, with a potential pro-tumorigenic role for OSM and pro-dormancy role for CNTF. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Suppression of autophagy by FIP200 deletion leads to osteopenia in mice through the inhibition of osteoblast terminal differentiation

Autophagy is a conserved lysosomal degradation process that has important roles in both normal human physiology and disease. However, the function of autophagy in bone homeostasis is not well understood. Here, we report that autophagy is activated during osteoblast differentiation. Ablation of focal adhesion kinase family interacting protein of 200 kD (FIP200), an essential component of mammalian autophagy, led to multiple autophagic defects in osteoblasts including aberrantly increased p62 expression, deficient LC3‐II conversion, defective autophagy flux, absence of GFP‐LC3 puncta in FIP200‐null osteoblasts expressing transgenic GFP‐LC3, and absence of autophagosome‐like structures by electron microscope examination. Osteoblast‐specific deletion of FIP200 led to osteopenia in mice. Histomorphometric analysis revealed that the osteopenia was the result of cell‐autonomous effects of FIP200 deletion on osteoblasts. FIP200 deletion led to defective osteoblast terminal differentiation in both primary bone marrow and calvarial osteoblasts in vitro. Interestingly, both proliferation and differentiation were not adversely affected by FIP200 deletion in early cultures. However, FIP200 deletion led to defective osteoblast nodule formation after initial proliferation and differentiation. Furthermore, treatment with autophagy inhibitors recapitulated the effects of FIP200 deletion on osteoblast differentiation. Taken together, these data identify FIP200 as an important regulator of bone development and reveal a novel role of autophagy in osteoblast function through its positive role in supporting osteoblast nodule formation and differentiation. © 2013 American Society for Bone and Mineral Research.     

IL-6 negatively regulates osteoblast differentiation through the SHP2/MEK2 and SHP2/Akt2 pathways in vitro

It has been suggested that interleukin-6 (IL-6) plays a key role in the pathogenesis of rheumatoid arthritis (RA), including osteoporosis not only in inflamed joints but also in the whole body. However, previous in vitro studies regarding the effects of IL-6 on osteoblast differentiation are inconsistent. The aim of this study was to examine the effects and signal transduction of IL-6 on osteoblast differentiation in MC3T3-E1 cells and primary murine calvarial osteoblasts. IL-6 and its soluble receptor significantly reduced alkaline phosphatase (ALP) activity, the expression of osteoblastic genes (Runx2, osterix, and osteocalcin), and mineralization in a dose-dependent manner, which indicates negative effects of IL-6 on osteoblast differentiation. Signal transduction studies demonstrated that IL-6 activated not only two major signaling pathways, SHP2/MEK/ERK and JAK/STAT3, but also the SHP2/PI3K/Akt2 signaling pathway. The negative effect of IL-6 on osteoblast differentiation was restored by inhibition of MEK as well as PI3K, while it was enhanced by inhibition of STAT3. Knockdown of MEK2 and Akt2 transfected with siRNA enhanced ALP activity and gene expression of Runx2. These results indicate that IL-6 negatively regulates osteoblast differentiation through SHP2/MEK2/ERK and SHP2/PI3K/Akt2 pathways, while affecting it positively through JAK/STAT3. Inhibition of MEK2 and Akt2 signaling in osteoblasts might be of potential use in the treatment of osteoporosis in RA.     

Differentiation in medulloblastomas and other primitive neuroectodermal tumours

Fifty-four cases of primitive neuroectodermal tumour (PNET) including 47 medulloblastomas, were examined for evidence of neuronal and glial differentiation, using antibodies to neuron-specific enolase (NSE), neurofilament protein (NF), and glial fibrillary acidic protein (GFAP). In 30 of the cases, antibodies to vimentin, alphafetoprotein, cytokeratin, epithelial membrane antigen and lymphoid markers were also used. Most of the 47 medulloblastomas in the group were NSE positive but NF negative; about half were GFAP positive and three of them were positive for both neuronal markers and for GFAP. Vimentin was demonstrated in four cases and was not always co-expressed with GFAP. Medulloblastomas were negative for all the other markers. Supratentorial PNETs were sometimes positive with neuronal markers but were GFAP negative. The cell specificity of these markers and the interpretation of immunocytochemical findings are discussed in relation to differentiation potential in primitive neuroectodermal tumours.     

高糖环境LIF介导STAT3/SOCS3信号通路参与成骨细胞分化机制研究

目的研究高糖环境下白血病抑制因子(leukemia inhibitory factor,LIF)介导STAT3/SOCS3信号通路对成骨细胞分化的影响及其作用机制。方法利用ELISA检测2型糖尿病患者血清白细胞介素6(interleukin-6,IL-6)家族中LIF及炎性因子的水平;通过RT-PCR、Western blot检测体外高糖环境下成骨分化标志物ALP、RUNX2、OCN、OPN以及细胞因子信号抑制因子3(suppressors of cytokine signaling,SOCS3)的m RNA和蛋白表达情况。结果 2型糖尿病患者血清炎性因子和LIF的水平明显升高。高糖或LIF显著降低了成骨分化标志物ALP、RUNX2、OCN和OPN的m RNA和蛋白水平,同时上调了SOCS 3和PSTAT3的表达。加入50 nmol/L或100 nmol/L STAT 3抑制剂JSI-124可改善成骨分化标志物的表达下调。结论 LIF通过介导STAT3/SOCS3信号通路参与高糖诱导下的成骨细胞分化。本研究为高糖导致的成骨障碍奠定了分子生物学理论基础并对靶点药物的研发提供了实验依据。     

Role of mitogen‐activated protein kinase in osteoblast differentiation

Local control of osteoblast differentiation and bone formation is not well understood. We have previously seen biphasic effects on cell differentiation in response to the short‐ and long‐term exposure to IL‐1β in rat calvarial osteoblasts. To characterize the signaling pathway mechanisms regulating IL‐1β biphasic effects, we examined the contribution of mitogen‐activated protein kinase (MAPK) family. Cells were pretreated with specific inhibitors to extracellular signal‐regulated kinase (ERK, PD98059), p38 (SB203580), and c‐JUN N‐terminal kinase (JNK, SP600125), then co‐cultured with IL‐1β for 2, 4, and 6 days. Cell differentiation was determined by measuring bone nodules after 10 days of culture. These inhibitors did not alter biphasic effects of IL‐1β on cell differentiation. However, PD98059 and U2016, another inhibitor of ERK activation robustly increased osteoblast differentiation compared to vehicle‐treated control in a time‐ and dose‐dependent manner. PD98059 appears to stimulate alkaline phosphatase (ALP) activity to promote cell differentiation, where IL‐1β appears to suppress it. Interestingly, continuous ERK inhibition with PD98059, after 2 and 4 days of IL‐1β treatment, enhanced the IL‐1β anabolic effect by increasing bone nodules formed. These observations provide a potential mechanism involving ERK pathway in osteoblasts differentiation and suggest that MAPK family may not directly regulate IL‐1β biphasic effects. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:204–210, 2011     

A selective EP4 receptor antagonist abrogates the stimulation of osteoblast recruitment from bone marrow stromal cells by prostaglandin E2 in vivo and in vitro

Recent evidence indicates that systemic administration of PGE2 increases bone formation and bone mass via activation of the EP4 receptor. Previously, we demonstrated that osteoblastic recruitment from rat bone marrow stromal cells (BMSC) is a major mechanism for the anabolic effect of PGE2. In this study, we used a selective EP4 antagonist to test if the stimulation of osteoblast differentiation from rat BMSC in vitro and in vivo involves the EP4 receptor. In vitro, PGE2 (100 nM) increased nodule formation and alkaline phosphatase (ALP) activity in cultures of rat BMSC 1.5- to 2-fold. These effects were abolished by the EP4 antagonist at 10(-6) M but not 10(-9) M. Furthermore, PGE2 increased the number of surviving adherent BMSC by approximately 225% and the EP4 antagonist prevented this effect as well. The antagonist had no effect on basal levels of nodule formation and adherent cell number. In vivo, daily systemic administration of PGE2 at 6 mg/kg for 2 weeks increased cancellous bone area (by approximately 50%) and increased nodule formation (measured as mineralized area) in ex vivo stromal cultures by approximately 50%. Pre-administration of the EP4 antagonist at 10 mg/kg abrogated both the increase in bone mass as well as the increase in nodule formation. These data indicate that PGE2 stimulates osteoblastic commitment of BMSC via activation of the EP4 receptor.     

Spectrum of osteoblastic differentiation in new cell lines derived from spontaneous murine osteosarcomas

Cell lines were established from three spontaneous osteosarcoma and one fibrosarcoma of aging mice. They were studied for tumorigenicity, osteoblastic features, and other in vitro cellular characteristics, by a combination of histological, morphological, biochemical, and molecular approaches. It was found that all cell lines formed tumors in vivo, whereas in vitro, only the fibrosarcoma-derived cell line grew efficiently in soft agar. Three out of the four cell lines produced mouse endogenous retroviruses, but none were classical sarcoma viruses. Type I collagen was expressed by all the cell lines, as was another extracellular matrix protein, osteonectin. The osteosarcoma-derived cell lines, however, exhibited different degrees of osteogenic differentiation. Only one line (OSA), and its clonal subline (1G11), consistently gave rise to mineralized tumors after transplantation into syngeneic mice, und these cells expressed high levels of alkaline phosphatase and bone-specific osteocalcin mRNA in vitro. Expression of these biochemical markers of osteoblasts occurred to a lesser extent in a second line (OSC) and was undetectable in the third line (OSB). The clonal 1G11 cell line exhibits the phenotype of a fully mature osteoblast and thus may serve as a particularly useful model for studies of bone cell function and regulation. Studies of cells which display a wide spectrum of osteogenic potential may further our understanding of the mechanisms involved in bone cell differentiation and tumorigenicity.     

Aging impairs IGF-I receptor activation and induces skeletal resistance to IGF-I.

IGF-I plays an important anabolic role in stimulating bone formation and maintaining bone mass. We show that the pro-proliferative, anti-apoptotic, and functional responses to IGF-I in bone and BMSCs decrease with aging. These changes are associated with impaired receptor activation and signal transduction through the MAPK and PI3K pathways. INTRODUCTION: IGF-I is a potent anabolic agent having effects across diverse tissues and cell types. With aging, bone becomes resistant to the anabolic actions of IGF-I. To examine the effects of aging on bone responsiveness to IGF-I, we measured the pro-proliferative, anti-apoptotic, and functional responses of bone and bone marrow stromal cells (BMSCs) to IGF-I and evaluated IGF-I signal transduction in young, adult, and old mice. MATERIALS AND METHODS: Male C57BL/6 mice 6 wk (young), 6 mo (adult), and 24 mo (old) were treated with IGF-I for 2 wk using osmotic minipumps, and osteoblast proliferation (BrdU labeling) in vivo, and osteoprogenitor number (BMSC culture and calcium nodule formation) were measured. Proliferation, apoptosis, and expression of key osteoblast factors (alkaline phosphatase, collagen, osteocalcin, RANKL, osteoprotegerin (OPG), macrophage-colony stimulating factor [M-CSF]) and IGF-I signaling elements and their activation in IGF-I-treated cells were studied using QRT-PCR and Western blot analysis. Data were analyzed using ANOVA. RESULTS: Aging decreased the basal and IGF-I-stimulated number of BrdU-labeled osteoblasts and reduced the ability of IGF-I to stimulate osteoprogenitor formation (calcium nodule number) by 50%. The pro-proliferative and anti-apoptotic actions of IGF-I were blunted in cells from old animals. These changes were accompanied by age-related alterations in the ability of IGF-I to regulate alkaline phosphatase, collagen, osteocalcin, RANKL, OPG, and M-CSF expression. IGF-I binding was normal, but IGF-I receptor mRNA and protein expression was increased in aged animals by 2- and 10-fold, respectively. The age-related changes in proliferation, apoptosis, and function were accompanied by loss of IGF-I-induced signaling at the receptor level and at key regulatory sites along the MAPK (ERK1/2) and PI3K (AKT) pathways. CONCLUSIONS: Our data show that aging is accompanied by loss of bone and BMSC/osteoblast responsiveness to IGF-I and that these changes are associated with resistance to IGF-I signaling that involve receptor activation and downstream signaling events.     

Down-Regulation of Osteoblastic Cell Differentiation by Epidermal Growth Factor Receptor

The role of epidermal growth factor receptors (EGF-R) in osteogenic cell differentiation was investigated using preosteoblastic MC3T3-E1 (MC3T3) cells and osteoblast-like ROS 17/2.8 (ROS) cells. When cultured in the presence of β-glycerophosphate (GP) and ascorbic acid (AA), MC3T3 cells underwent spontaneous differentiation into osteoblasts which was confirmed as they expressed osteoblast markers such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC). Interestingly, the number of EGF-binding sites decreased during their differentiation into osteoblasts, and the osteogenic protein-1 (OP-1) treatment, which accelerated their differentiation, lowered the number of EGF-binding sites even further. On the other hand, ROS cells with high expression levels of osteoblast markers and no EGF-R, after being transfected with human EGF-R cDNA (EROS cells), expressed numerous EGF-binding sites as well as EGF-R mRNA and protein; in the process, they ceased to express osteoblast markers, indicating their dedifferentiation into osteoprogenitor cells. Both MC3T3 and EROS cells showed increased cell growth in response to EGF, whereas ROS cells did not. These results imply that the EGF/EGF-R system in osteogenic cells has a crucial function in osteoblast phenotype suppression and osteogenic cell proliferation.     

Activation of Sirt1 decreases adipocyte formation during osteoblast differentiation of mesenchymal stem cells.

In vitro, mesenchymal stem cells differentiate to osteoblasts when exposed to bone-inducing medium. However, adipocytes are also formed. We showed that activation of the nuclear protein deacetylase Sirt1 reduces adipocyte formation and promotes osteoblast differentiation. INTRODUCTION: Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, adipocytes, chondrocytes, and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSCs into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor gamma2 (PPARgamma2) is a key element for the differentiation into adipocytes. Activation of Sirt1 has recently been shown to decrease adipocyte development from preadipocytes through inhibition of PPARgamma2. MATERIALS AND METHODS: We used the mouse mesenchymal cell line C3H10T1/2 and primary rat bone marrow cells cultured in osteoblast differentiation medium with or without reagents affecting Sirt1 activity. Adipocyte levels were analyzed by light microscopy and flow cytometry (FACS) after staining with Oil red O and Nile red, respectively. Osteoblast and adipocyte markers were studied with quantitative real-time PCR. Mineralization in cultures of primary rat bone marrow stromal cells was studied by von Kossa and alizarin red staining. RESULTS: We found that Sirt1 is expressed in the mesenchymal cell line C3H10T1/2. Treatment with the plant polyphenol resveratrol as well as isonicotinamide, both of which activate Sirt1, blocked adipocyte development and increased the expression of osteoblast markers. Nicotinamide, which inhibits Sirt1, increased adipocyte number and increased expression of adipocyte markers. Furthermore, activation of Sirt1 prevented the increase in adipocytes caused by the PPARgamma-agonist troglitazone. Finally, activation of Sirt1 in rat primary bone marrow stromal cells increased expression of osteoblast markers and also mineralization. CONCLUSIONS: In this study, we targeted Sirt1 to control adipocyte development during differentiation of MSCs into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation may be relevant in the search for new treatment regimens of osteoporosis but also important for the evolving field of cell-based tissue engineering.     

p21(WAF1/CIP1) acts as a brake in osteoblast differentiation.

Continuous fibroblast growth factor signaling inhibits the differentiation of primary osteoblasts and osteoblastic cell lines. We studied the expression of several cell cycle regulatory molecules in response to fibroblast growth factor, and found that fibroblast growth factor strongly upregulates the expression of p21(WAF1/CIP1), a CDK inhibitor that has also been implicated in the regulation of apoptosis and cell differentiation. To test the hypothesis that p21 mediated the fibroblast growth factor effects on osteoblasts, we studied the differentiation of primary osteoblasts and osteoblastic cell lines derived from p21 null mice in the presence or absence of fibroblast growth factor. While the results obtained indicate that p21 is not the major mediator of the inhibition of osteoblast differentiation by fibroblast growth factor, we found that p21 per se acts as a brake on osteoblast proliferation and differentiation. p21 is strongly downregulated during differentiation and is highly expressed in osteoblastic cell lines expressing activated FGFR2, which do not differentiate. p21 null osteoblasts differentiate faster than wild-type cells, are more susceptible to the differentiation-promoting action of BMP-2, and undergo increased differentiation-related apoptosis. Furthermore, transient overexpression of p21 from an adenovirus vector delayed the onset of differentiation both in wild-type and in p21 null osteoblasts. These results highlight a new function for p21 in osteoblast differentiation.     

Bioglass ®45S5 Stimulates Osteoblast Turnover and Enhances Bone Formation In Vitro: Implications and Applications for Bone Tissue Engineering

We investigated the concept of using bioactive substrates as templates for in vitro synthesis of bone tissue for transplantation by assessing the osteogenic potential of a melt-derived bioactive glass ceramic (Bioglass 45S5) in vitro. Bioactive glass ceramic and bioinert (plastic) substrates were seeded with human primary osteoblasts and evaluated after 2, 6, and 12 days. Flow cytometric analysis of the cell cycle suggested that the bioactive glass-ceramic substrate induced osteoblast proliferation, as indicated by increased cell populations in both S (DNA synthesis) and G2/M (mitosis) phases of the cell cycle. Biochemical analysis of the osteoblast differentiation markers alkaline phosphatase (ALP) and osteocalcin indicated that the bioactive glass-ceramic substrate augmented osteoblast commitment and selection of a mature osteoblastic phenotype. Scanning electron microscopic observations of discrete bone nodules over the surface of the bioactive material, from day 6 onward, further supported this notion. A combination of fluorescence, confocal, transmission electron microscopy, and X-ray microprobe (SEM-EDAX) examinations revealed that the nodules were made of cell aggregates which produced mineralized collagenous matrix. Control substrates did not exhibit mineralized nodule formation at any point studied up to 12 days. In conclusion, this study shows that Bioglass 45S5 has the ability to stimulate the growth and osteogenic differentiation of human primary osteoblasts. These findings have potential applications for tissue engineering where this bioactive glass substrate could be used as a template for the formation of bioengineered bone tissue.     

骨肉瘤中cbfa1基因表达与临床因素的相关性研究

目的 ｃｂｆａｌ基因是一种结合在Ｏｓｔｅｏｃａｌｃｉｎ启动子上并能调节其在成骨细胞中表达的转录因子,在成骨细胞的分化和成熟中起着重要的作用。由于ｃｂｆａｌ基因在成骨分化过程中所起的重要作用。研究骨肉瘤中ｃｂｆａｌ的表达有重要的意义。方法 应用Ｓｏｕｔｈｅｒｎ ｂｌｏｔ法ＲＴ－ＰＣＲ技术对４３例骨肉瘤标本和３株骨肉瘤细胞系（ＳＯＳ－２,ＨＯＳ和Ｕ－２）中ｃｂｆａｌ基因的结构和表达进行了研究。