Epidermal growth factor and calcitriol synergistically induce osteoblast maturation

Calcitriol (1,25(OH)₂D₃) plays a key role in the differentiation of osteoblasts, the cells responsible for the formation and maintenance of healthy bone matrix. Recently it has emerged that calcitriol influences the trafficking or stability of epidermal growth factor (EGF) receptors. However, how these agents might work together in regulating growth and differentiation has not been examined. Using the human osteoblast cell line, MG63, we were able to induce a profound differentiation response by treating these cells with a combination of calcitriol (100 nM) and EGF (10 ng/ml). Co-stimulation of MG63 osteoblasts with calcitriol and EGF led to synergistic increases in osteocalcin and alkaline phosphatase (ALP), proteins expressed by differentiating cells. Inhibition of differentiation was accomplished by MEK and protein kinase C (PKC) inhibitors. Other ligands known to signal via receptor tyrosine kinases could not substitute for EGF in the maturation response. These novel findings may help identify new processes that drive osteoblast differentiation.     

Aging compromises oligodendrocyte precursor cell maturation and efficient remyelination in the monkey brain

GeroScience - Age-associated cognitive decline is common among otherwise healthy elderly people, even in the absence of Alzheimer’s disease and neuron loss. Instead, white matter loss and...     

Autocrine signals promote osteoblast survival in culture

We have studied the survival requirements of osteoblasts to test the hypothesis that osteoblasts undergo programmed cell death (PCD) or apoptosis unless they are continuously signalled by other cells not to do so. Osteoblasts survived for 6 days in culture at high cell density in the absence of other cell types, serum or exogenous proteins, but they died with the morphological features of apoptosis in these conditions at low cell density. Osteoblast survival was enhanced during the first 2 days of culture by the addition of the sulphydryl compound, cysteine to the culture medium which was converted intracellularly to the antioxidant glutathione. Catalase, an enzyme decomposing hydrogen peroxide, also protected the cells, whereas superoxide dismutase had no effect. Therefore, osteoblasts in culture are sensitive to toxic compounds derived from molecular oxygen, i.e. hydroxyl radicals or hydrogen peroxide spontaneously generated in CMRL medium containing ascorbate and ferrous ions. Conditioned medium from high density cultures prevented osteoblast apoptosis in low density cultures, as long as antioxidants were also present. The enhancing effect of conditioned medium on osteoblast survival was prevented by neutralizing antibodies to insulin-like growth factor-I (IGF-I) and IGF-II but not by antibodies to either platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF). These results suggest that in addition to regulating cell growth and differentiation, IGF-I and IGF-II also function as survival factors for osteoblasts. Our data also indicate that antioxidants are required for osteoblast survival and that they enhance growth factor mediated osteoblast survival.     

SHIP deficiency enhances HSC proliferation and survival but compromises homing and repopulation

The SH2 domain-containing inositol 5'-phosphatase-1 (SHIP) has the potential to modulate multiple signaling pathways downstream of receptors that impact hematopoietic stem cell (HSC) biology. Therefore, we postulated that SHIP might play an important role in HSC homeostasis and function. Consistent with this hypothesis, HSC proliferation and numbers are increased in SHIP(-/-) mice. Despite expansion of the compartment, SHIP(-/-) HSCs exhibit reduced capacity for long-term repopulation. Interestingly, we observe that SHIP(-/-) stem/progenitor cells home inefficiently to bone marrow (BM), and consistent with this finding, have reduced surface levels of both CXCR4 and vascular cell adhesion marker-1 (VCAM-1). These studies demonstrate that SHIP is critical for normal HSC function, homeostasis, and homing.     

The BCR-ABLT315I mutation compromises survival in chronic phase chronic myelogenous leukemia patients resistant to tyrosine kinase inhibitors,in a matched pair analysis

The BCR-ABL T315I mutation confers resistance to currently licensed tyrosine kinase inhibitors in chronic myelogenous leukemia. However, the impact of this mutation on survival in early stages of disease, in chronic phase, has never been detailed. Using matched pair analysis, a cohort of 64 patients with chronic phase chronic myelogenous leukemia harboring a T315I mutation and resistant to imatinib mesylate was compared to a similar cohort of 53 chronic phase patients resistant to imatinib, but with no detectable T315I mutation, in the pre-ponatinib era. These patients were matched according to age at diagnosis, interval between disease diagnosis and start of imatinib treatment, and duration of imatinib therapy. Kaplan-Meier survival analyses demonstrated the significant negative impact of the presence of the T315I mutation on overall survival (since imatinib-resistance: 48.4 months for T315I⁺ patients versus not reached for T315I⁻ ones; P=0.006) and failure-free survival (since imatinib-resistance: 34.7 months for T315I⁺ patients versus not reached for T315I⁻ patients; P=0.003). In addition, Cox proportional hazard models adjusted on overall survival demonstrated the negative influence of the T315I mutation (P=0.02, HR=2.54). These results confirm early assumptions concerning the poor prognosis of chronic phase chronic myelogenous leukemia patients with the T315I mutation who are not eligible for allogeneic transplantation, and demonstrate the need for more therapeutic options.     

Calcineurin regulates bone formation by the osteoblast

Two of the most commonly used immunosuppressants, cyclosporine A and tacrolimus (FK506), inhibit the activity of a ubiquitously expressed Ca(2+)/calmodulin-sensitive phosphatase, calcineurin. Because both drugs also cause profound bone loss in humans and in animal models, we explored whether calcineurin played a role in regulating skeletal remodeling. We found that osteoblasts contained mRNA and protein for all isoforms of calcineurin A and B. TAT-assisted transduction of fusion protein TAT-calcineurin Aalpha into osteoblasts resulted in the enhanced expression of the osteoblast differentiation markers Runx-2, alkaline phosphatase, bone sialoprotein, and osteocalcin. This expression was associated with a dramatic enhancement of bone formation in intact calvarial cultures. Calcineurin Aalpha(-/-) mice displayed severe osteoporosis, markedly reduced mineral apposition rates, and attenuated colony formation in 10-day ex vivo stromal cell cultures. The latter was associated with significant reductions in Runx2, bone sialoprotein, and osteocalcin expression, paralleled by similar decreases in response to FK506. Together, the gain- and loss-of-function experiments indicate that calcineurin regulates bone formation through an effect on osteoblast differentiation.     

Insulin-like growth factor-binding protein 5 (Igfbp5) compromises survival, growth, muscle development, and fertility in mice

The insulin-like growth factors (IGFs) are essential for development; bioavailable IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). Igfbp5 is the most conserved and is developmentally up-regulated in key lineages and pathologies; in vitro studies suggest that IGFBP-5 functions independently of IGF interaction. Genetic ablation of individual Igfbps has yielded limited phenotypes because of substantial compensation by remaining family members. Therefore, to reveal Igfbp5 actions in vivo, we generated lines of transgenic mice that ubiquitously overexpressed Igfbp5 from early development. Significantly increased neonatal mortality, reduced female fertility, whole-body growth inhibition, and retarded muscle development were observed in Igfbp5-overexpressing mice. The magnitude of the response in individual transgenic lines was positively correlated with Igfbp5 expression. Circulating IGFBP-5 concentrations increased a maximum of only 4-fold, total and free IGF-I concentrations increased up to 2-fold, and IGFBP-5 was detected in high M(r) complexes; however, no detectable decrease in the proportion of free IGF-I was observed. Thus, despite only modest changes in IGF and IGFBP concentrations, the Igfbp5-overexpressing mice displayed a phenotype more extreme than that observed for other Igfbp genetic models. Although growth retardation was obvious prenatally, maximal inhibition occurred postnatally before the onset of growth hormone-dependent growth, regardless of Igfbp5 expression level, revealing a period of sensitivity to IGFBP-5 during this important stage of tissue programming.     

Molecular mechanisms of cartilage formation and chondrocyte maturation

Cartilage plays multiple roles in vertebrate animals. In an embryonic stage and early postnatal life, cartilage is important not only as a structural support of early embryo but also as a template of endochondral bone. In a later postnatal life, cartilage provides smooth joint movement and tissue elasticity. A number of critical signaling molecules that regulate cartilage formation and chondrocytes maturation in endochondral bone formation have been identified to date. The interplay of those important molecules is also actively studied. However, several fundamental questions still remain unsolved. What signal initiates mesenchymal cell condensation? Does condensation enough to make cells competent for BMP-induced chondrogenesis? Is there chondrocyte stem cell in cartilage? Likewise, it is not known which factor triggers chondrocytes maturation. In this review article, we summarized the action of several key factors including BMP, hedgehog, PTHrP, and Wnt in condensation, chondrogenenic differentiation and maturation of chondrocytes. Towards further understanding of above fundamental questions, this review article also tried to propose future direction of cartilage biology research.     

氟剂对成骨细胞成骨功能表达的影响

目的　从不同剂量氟化钠 (NaF)对大鼠成骨细胞 (OB)功能表达的影响 ,研究氟剂促进成骨作用的机制及剂量效应关系。方法　经酶消化法从新生 2 4hSpregne Dawley (SD)种乳鼠头盖骨分离得到的成骨细胞。在 10 -7～ 5× 10 -4mol/LNaF中培养。用生化法测定细胞碱性磷酸酶 (ALP)活性 ,用放射免疫法测定培养液中骨钙素 (OC)含量。结果　NaF对细胞ALP活性的影响呈双向 ,即低浓度NaF(10 -7～ 10 -5mol/L)增加ALP活性 ,高浓度NaF (10 -4～ 5× 10 -4mol/L)则抑制ALP活性 ;对骨钙素产生和分泌的影响则呈单向 ,即各浓度NaF均刺激细胞骨钙素分泌 ,并与氟剂剂量呈正相关 ,其中 5× 10 -4mol/L组骨钙素水平最高 ,与对照组相比增加 6 7 14 % (P <0 0 5 )。结论　适量氟剂能增加成骨细胞ALP的活性及骨钙素的产生 ,促进成骨细胞的骨形成功能 ,但有效剂量范围狭窄     

Culture in vitro of isolated guinea pig megakaryocytes: recovery, survival, morphologic changes, and maturation

Isolated guinea pig megakaryocytes were maintained in liquid cultures for up to 4 days. Megakaryocytes were incubated in siliconized glass vials in Dulbecco's Modified Eagle Medium with 5%-10% guinea pig serum and 2.3% bovine serum albumin. Cultured megakaryocytes did not adhere to glass vials and were almost entirely recovered by aspiration. No reproduction or cell division of megakaryocytes occurred. A small decline in viability occurred promptly on placing the freshly isolated cells in culture medium and could be attributed to reexposure to calcium. On incubation there was little further cell death. Up to 2 days in culture the megakaryocytes remained morphologically intact and appeared similar to megakaryocytes in situ. Megakaryocytes matured in culture with a loss of cytoplasmic basophilia, an increase in granule content, and progressive changes in nuclear configuration. The most mature megakaryocytes developed pseudopod formation but large-scale platelet liberation was not seen. The ability to culture megakaryocytes in vitro will allow more extensive biochemical and physiologic studies of this cell than previously possible.     

Loss of Tie2 receptor compromises embryonic stem cell-derived endothelial but not hematopoietic cell survival

Tie2 is a receptor-type tyrosine kinase expressed on hematopoietic stem cells and endothelial cells. We used cultured embryonic stem (ES) cells to determine the function of Tie2 during early vascular development and hematopoiesis. Upon differentiation, the ES cell-derived Tie2+ Flk1+ fraction was enriched for hematopoietic and endothelial progenitor cells. To investigate lymphatic differentiation, we used a monoclonal antibody against LYVE-1 and found that LYVE-1+ cells derived from Tie2+ Flk1+ cells possessed various characteristics of lymphatic endothelial cells. To determine whether Tie2 played a role in this process, we analyzed differentiation of Tie2-/- ES cells. Although the initial numbers of LYVE-1+ and PECAM-1+ cells derived from Tie2-/- cells did not vary significantly, the number of both decreased dramatically upon extended culturing. Such decreases were rescued by treatment with a caspase inhibitor, suggesting that reductions were due to apoptosis as a consequence of a lack of Tie2 signaling. Interestingly, Tie2-/- ES cells did not show measurable defects in development of the hematopoietic system, suggesting that Tie2 is not essential for hematopoietic cell development.     

Inherited thrombocytopenia: novel insights into megakaryocyte maturation,proplatelet formation and platelet lifespan

The study of patients with inherited bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets and their precursor, the megakaryocyte. The normal range of platelet counts in the bloodstream ranges from 150 000 to 400 000 platelets per microliter and is normally maintained within a narrow range for each individual. This requires a constant balance between thrombopoiesis, which is primarily controlled by the cytokine thrombopoietin (TPO), and platelet senescence and consumption. Thrombocytopenia can be defined as a platelet count of less than 150 000 per microliter and can be acquired or inherited. Heritable forms of thrombocytopenia are caused by mutations in genes involved in megakaryocyte differentiation, platelet production and platelet removal. In this review, we will discuss the main causative genes known for inherited thrombocytopenia and highlight their diverse functions and whether these give clues on the processes of platelet production, platelet function and platelet lifespan. Additionally, we will highlight the recent advances in novel genes identified for inherited thrombocytopenia and their suggested function.     

Ovariectomy disregulates osteoblast and osteoclast formation through the T-cell receptor CD40 ligand

The bone loss induced by ovariectomy (ovx) has been linked to increased production of osteoclastogenic cytokines by bone marrow cells, including T cells and stromal cells (SCs). It is presently unknown whether regulatory interactions between these lineages contribute to the effects of ovx in bone, however. Here, we show that the T-cell costimulatory molecule CD40 ligand (CD40L) is required for ovx to expand SCs; promote osteoblast proliferation and differentiation; regulate the SC production of the osteoclastogenic factors macrophage colony-stimulating factor, receptor activator of nuclear factor-κB ligand, and osteoprotegerin; and up-regulate osteoclast formation. CD40L is also required for ovx to activate T cells and stimulate their production of TNF. Accordingly, ovx fails to promote bone loss and increase bone resorption in mice depleted of T cells or lacking CD40L. Therefore, cross-talk between T cells and SCs mediated by CD40L plays a pivotal role in the disregulation of osteoblastogenesis and osteoclastogenesis induced by ovx.     

前列腺素E2促进老年大鼠成骨细胞骨形成及其机制研究

目的　探讨前列腺素E2 (PGE2 )对老年雄性大鼠胫骨上段松质骨的作用。　方法　取2 0月龄Wistar雄性大鼠 ,分别皮下注射PGE2 (3mg·kg 1·d 1) 10d和 30d。用体内双荧光标记法、不脱钙组织切片 ,以骨组织形态计量学方法观察胫骨上段松质骨骨小梁和骨髓腔的动态和静态参数变化。　结果　PGE2 作用 10d骨小梁表面成骨细胞周长OB/BS〔(12 3± 7 6 ) %〕和类骨质周长OS/BS〔(2 0 4± 7 2 ) %〕明显高于对照组〔(1 6± 0 7) %和 (4 3± 1 7) % ,P <0 0 1〕 ,骨小梁表面和髓腔出现多层排列的前成骨细胞OPC ,形成多细胞成分而钙化不全的新生骨小梁WB ;PGE2 作用 30d矿化骨形成率BFR/BV〔(815 4± 137 9) %·年 1〕和骨量BV/TV〔(4 2 1± 12 6 ) %〕明显高于对照组〔(15 4 9± 14 6 5 ) %·年 1和 (13 5± 3 2 ) % ,P <0 0 1〕 ,骨髓脂肪组织面积F/TV由 (18 2± 5 6 ) %减少到 (11 4± 3 6 ) % (P <0 0 5 )。　结论　PGE2 在短期内有刺激老年大鼠松质骨成骨细胞骨形成 ,增加骨量的作用。     

Pregnancy-associated plasma protein-A increases osteoblast proliferation in vitro and bone formation in vivo

Pregnancy-associated plasma protein (PAPP)-A, a protease for IGF binding protein (IGFBP)-2, -4, and -5, may enhance IGF action by increasing its bioavailability. Here we have determined the role and mechanism of action of PAPP-A in the regulation of osteoblast proliferation in vitro and bone metabolism in vivo. Recombinant PAPP-A (100 ng/ml) significantly increased osteoblast proliferation and free IGF-I concentration. These effects were abolished by noncleavable IGFBP-4, suggesting that PAPP-A promotes osteoblast proliferation by increasing IGF bioavailability. To determine whether PAPP-A exerts an anabolic effect on bone in vivo, we developed transgenic mice that overexpress PAPP-A in osteoblasts using the 2.3-kb rat type I collagen promoter. Consistent with the increase in IGFBP-4 proteolysis, free IGF-I concentration was significantly increased in the conditioned medium of cultured osteoblasts derived from transgenic mice compared with the wild-type littermates. Calvarial bone thickness, bone marrow cavity, and skull bone mineral density were significantly increased in transgenic mice. Bone size-related parameters in femur and tibia such as total bone area and periosteal circumference as determined by peripheral quantitated computed tomography and histological analysis were significantly increased in transgenic mice. Bone formation rate and osteoid surface were increased by more than 2-fold, whereas bone resorbing surface was unaffected. These anabolic effects were sustained with aging. These findings provide strong evidence that PAPP-A acts as a potent anabolic factor in the regulation of bone formation. Thus, enhancing IGF bioavailability by PAPP-A can be a powerful strategy in the treatment of certain metabolic diseases such as osteoporosis.