Ubiquitin C-terminal hydrolase-L3 regulates Smad1 ubiquitination and osteoblast differentiation

Ubiquitin C-terminal hydrolase-L3 (Uch-L3), a deubiquitinating enzyme, is upregulated in bone morphogenetic protein 2-induced osteoblast differentiation. The mechanism and role of Uch-L3 in the process of osteoblast differentiation is unknown. We found that Uch-L3 physically interacts with Smad1 and dramatically decreases the amount of poly-ubiquitinated Smad1. Osteoblast differentiation was enhanced in the C2C12 cells stably transfected with Uch-L3. Otherwise, the siRNA knock-down of Uch-L3 resulted in the decrease of osteoblast differentiation. These results suggest that Uch-L3 enhances osteoblast differentiation through the stabilization of Smad1 signaling. Thus, Uch-L3 acts to fine-tune the process of Smad1 activation.     

Post-translational modifications of collagen upon BMP-induced osteoblast differentiation

The pattern of collagen cross-linking is tissue specific primarily determined by the extent of hydroxylation and oxidation of specific lysine residues in the molecule. In this study, murine pre-myoblast cell line, C2C12 cells, were transdifferentiated into osteoblastic cells by bone morphogenetic protein (BMP)-2 treatment, and the gene expression of lysyl hydroxylases (LH1, 2a/b, and 3) and lysyl oxidase (LOX)/lysyl oxidase-like proteins (LOXL1-4), and the extent of hydroxylysine were analyzed. After 24 h of treatment, the expression of most isoforms were upregulated up to 96 h whereas LH2a and LOXL2 decreased with time. In the treated cells, both hydroxyproline and hydroxylysine were detected at day 7 and increased at day 14. The ratio of hydroxylysine to hydroxyproline was significantly increased at day 14. The results indicate that LHs and LOX/LOXLs are differentially responsive to BMP-induced osteoblast differentiation that may eventually lead to the specific collagen cross-linking pattern seen in bone.     

Thinning affects Pinus sylvestris needle decomposition rates and chemistry differently depending on site conditions

Changes in mass and chemical composition of Pinus sylvestris senescent needles were studied over a 5 year period in Mediterranean (MF) and Continental forests (CF) in the Pyrenees under varying levels of thinning (P0: reference, no thinning; P20: removal of 20% basal area, P30: removal of 30% basal area). Decomposition rates were higher in MF (k = 0.423 year^?1) than in CF (k = 0.245 year^?1). However, the maximum decomposition limit was higher in MF (87.9%) compared to CF (78.1%). The relative importance and timing of rainfall, and cellulose and lignin abundance on the decomposition process was similar among both sites. However, air temperature and degree-days only affected CF (the colder site) during the initial stages of decomposition, while litter moisture was significant only in MF (the drier site) in the latter stages of decomposition. Nutrient and carbon dynamics showed temporal patterns similar to those reported in higher latitudes (except for Ca), however, indicators of N mineralization such as C/N and lignin/N at the study sites were lower than values reported in the literature. Decreases in decomposition rates after thinning were higher in MF than in CF, indicating that this ecosystem could, in the short term, be more sensitive to human intervention. Thinning had similar temporary qualitative effects at both sites, slowing decomposition, increasing N and P immobilization and decreasing Ca immobilization. However, quantitative effects of thinning were site dependent in that the magnitude of nutrient immobilization was higher in CF. A conceptual model is presented to explain effects of thinning on litter N dynamics. These temporary changes are not trivial as nutrient immobilization and accumulated organic matter losses over a thinning cycle may affect tree growth particularly during short rotations and intensive fast-growing plantations. Under similar nutrient availability conditions, sites where nutrient release occurs faster may show higher post-thinning tree growth rates.     

Galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway

Galectin-9 is a β-galactoside-binding lectin expressed in various tissues. It binds various glycoconjugates and modulates a variety of biological functions in various cell types. Although galectin-9 is expressed in bone, its function in human osteoblasts remains unclear. We demonstrate that galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway in the absence of bone morphogenetic proteins (BMPs). Galectin-9 increases alkaline phosphatase activities in human osteoblasts and induces the phosphorylation of Smad1/5/8 and translocation of Smad4 to the nucleus in the absence of BMPs. Galectin-9 also induces binding of Smad4 to the Id1 promoter and increases its activity. Anti-CD44 antibody inhibits Smad1/5/8 phosphorylation by galectin-9. Galectin-9 binds to CD44 and induces the formation of a CD44/BMP receptor complex. Because Smad1 is phosphorylated by BMP receptors, we propose that formation of the CD44/BMP receptor complex induced by galectin-9 may provide a trigger for the activation of Smads.     

The effects of BIG-3 on osteoblast differentiation are not dependent upon endogenously produced BMPs

BMPs play an important role in both intramembranous and endochondral ossification. BIG-3, BMP-2-induced gene 3 kb, encodes a WD-40 repeat protein that accelerates the program of osteoblastic differentiation in vitro. To examine the potential interactions between BIG-3 and the BMP-2 pathway during osteoblastic differentiation, MC3T3-E1 cells stably transfected with BIG-3 (MC3T3E1-BIG-3), or with the empty vector (MC3T3E1-EV), were treated with noggin. Noggin treatment of pooled MC3T3E1-EV clones inhibited the differentiation-dependent increase in AP activity observed in the untreated MC3T3E1-EV clones but did not affect the increase in AP activity in the MC3T3E1-BIG-3 clones. Noggin treatment decreased the expression of Runx2 and type I collagen mRNAs and impaired mineralized matrix formation in MC3T3E1-EV clones but not in MC3T3E1-BIG-3 clones. To determine whether the actions of BIG-3 on osteoblast differentiation converged upon the BMP pathway or involved an alternate signaling pathway, Smad1 phosphorylation was examined. Basal phosphorylation of Smad1 was not altered in the MC3T3E1-BIG-3 clones. However, these clones did not exhibit the noggin-dependent decrease in phosphoSmad1 observed in the MC3T3E1-EV clones, nor did it decrease nuclear localization of phosphoSmad1. These observations suggest that BIG-3 accelerates osteoblast differentiation in MC3T3-E1 cells by inducing phosphorylation and nuclear translocation of Smad1 independently of endogenously produced BMPs.     

Human monocyte differentiation stage affects response to arachidonic acid

AA-induced cell death mechanisms acting on human monocytes and monocyte-derived macrophages (MDM), U937 promonocytes and PMA-differentiated U937 cells were studied. Arachidonic acid induced apoptosis and necrosis in monocytes and U937 cells but only apoptosis in MDM and U937D cells. AA increased both types of death in Mycobacterium tuberculosis-infected cells and increased the percentage of TNFα+ cells and reduced IL-10+ cells. Experiments blocking these cytokines indicated that AA-mediated death was TNFα- and IL-10-independent. The differences in AA-mediated cell death could be explained by high ROS, calpain and sPLA-2 production and activity in monocytes. Blocking sPLA-2 in monocytes and treatment with antioxidants favored M. tuberculosis control whereas AA enhanced M. tuberculosis growth in MDM. Such evidence suggested that AA-modulated effector mechanisms depend on mononuclear phagocytes’ differentiation stage.     

Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation

The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.     

Chondroitin sulfate-E fine-tunes osteoblast differentiation via ERK1/2, Smad3 and Smad1/5/8 signaling by binding to N-cadherin and cadherin-11

Bone formation in the vertebrate skeleton occurs via the processes of endochondral and membranous ossification. Bone matrices contain chondroitin sulfate (CS) chains that regulate endochondral ossification. However, the function of CS in membranous ossification is unclear. Here, using preosteoblastic MC3T3-E1 cells we demonstrate that chondroitin sulfate-E (CS-E) promotes osteoblast differentiation by binding to both N-cadherin and cadherin-11. Differentiated MC3T3-E1 cells exhibited an increase in the total amount of CS and of E-disaccharide units of CS over time. In addition, CS-E polysaccharide, but not CS-A polysaccharide, bound to N-cadherin and cadherin-11 and enhanced osteoblast differentiation. In contrast, osteoblast differentiation was inhibited in chondroitinase ABC-digested MC3T3-E1 cells. Notably, CS-E polysaccharide and hexasaccharide activated intracellular signaling during osteoblast differentiation in non-contacting MC3T3-E1 cells, decreased ERK1/2 phosphorylation, and activated Smad3 and Smad1/5/8; these reactions were blocked by neutralizing antibodies against N-cadherin or cadherin-11, even though cell-cell adhesion is reported to be required for initiation of MC3T3-E1 cell differentiation. Furthermore, CS-E-unit overexpression in MC3T3-E1 cells increased adhesion of the cells to N-cadherin and cadherin-11, and promoted osteoblast differentiation. Collectively, these results suggest that CS-E is a selective ligand for the potential CS receptors, N-cadherin and cadherin-11, leading to osteoblast differentiation of MC3T3-E1 cells.     

Parathyroid hormone-responsive Smad3-related factor, Tmem119, promotes osteoblast differentiation and interacts with the bone morphogenetic protein-Runx2 pathway

The mechanisms whereby the parathyroid hormone (PTH) exerts its anabolic action on bone are incompletely understood. We previously showed that inhibition of ERK1/2 enhanced Smad3-induced bone anabolic action in osteoblasts. These findings suggested the hypothesis that changes in gene expression associated with the altered Smad3-induced signaling brought about by an ERK1/2 inhibitor would identify novel bone anabolic factors in osteoblasts. We therefore performed a comparative DNA microarray analysis between empty vector-transfected mouse osteoblastic MC3T3-E1 cells and PD98059-treated stable Smad3-overexpressing MC3T3-E1 cells. Among the novel factors, Tmem119 was selected on the basis of its rapid induction by PTH independent of later increases in endogenous TGF-β. The levels of Tmem119 increased with time in cultures of MC3T3-E1 cells and mouse mesenchymal ST-2 cells committed to the osteoblast lineage by BMP-2. PTH stimulated Tmem119 levels within 1 h as determined by Western blot analysis and immunocytochemistry in MC3T3-E1 cells. MC3T3-E1 cells stably overexpressing Tmem119 exhibited elevated levels of Runx2, osteocalcin, alkaline phosphatase, and β-catenin, whereas Tmem119 augmented BMP-2-induced Runx2 levels in mesenchymal cells. Tmem119 interacted with Runx2, Smad1, and Smad5 in C2C12 cells. In conclusion, we identified a Smad3-related factor, Tmem119, that is induced by PTH and promotes differentiation in mouse osteoblastic cells. Tmem119 is an important molecule in the pathway downstream of PTH and Smad3 signaling in osteoblasts.     

Mycophenolic acid differentially impacts B cell function depending on the stage of differentiation

Production of pathogenic Abs contributes to disease progression in many autoimmune disorders. The immunosuppressant agent mycophenolic acid (MPA) has shown clinical efficacy for patients with autoimmunity. The goal of these studies was to elucidate the mechanisms of action of MPA on B cells isolated from healthy individuals and autoimmune patients. In this study, we show that MPA significantly inhibited both proliferation and differentiation of primary human B cells stimulated under various conditions. Importantly, MPA did not globally suppress B cell responsiveness or simply induce cell death, but rather selectively inhibited early activation events and arrested cells in the G0/G1 phase of the cell cycle. Furthermore, MPA blocked expansion of both naive and memory B cells and prevented plasma cell (PC) differentiation and Ab production from healthy controls and individuals with rheumatoid arthritis. Finally, whereas MPA potently suppressed Ig secretion from activated primary B cells, terminally differentiated PCs were not susceptible to inhibition by MPA. The target of MPA, IMPDH2, was found to be downregulated in PCs, likely explaining the resistance of these cells to MPA. These results suggest that MPA provides benefit in settings of autoimmunity by directly preventing activation and PC differentiation of B cells; however, MPA is unlikely to impact autoantibody production by preexisting, long-lived PCs.     

Chondrogenic differentiation in chick embryo osteoblast cultures.

Expression of specific differentiation markers was investigated by histochemistry, immunofluorescence, and biosynthetic studies in osteoblasts outgrown from chips derived from tibia diaphyses of 18-day-old chick embryos. The starting osteoblast population expressed type I collagen and alkaline phosphatase in addition to other bone and cartilage markers as the lipocalin Ch21; the extracellular matrix deposited by these cells was not stainable for cartilage proteoglycans, and mineralization was observed when the culture was maintained in the presence of ascorbic acid, calcium and beta-glycerophosphate. During culture, clones of cells presenting a polygonal chondrocyte morphology and surrounded by an Alcian-positive matrix appeared in the cell population. Type II collagen and type X collagen were synthesized in these areas of chondrogenesis. In addition, chondrocytes isolated from these cultures expressed Ch21 and alkaline phosphatase. Chondrocytes were generated also from homogeneous osteoblast populations derived from a single cloned cell. The coexistence of chondrocytes and osteoblasts was observed during amplification of primary clones as well as in subclones. The data show the existence, within embryonic bone, of cells capable in vitro of both osteogenic and chondrogenic differentiation.     

Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation — a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.