SVEP1 expression is regulated in estrogen-dependent manner

The SVEP1 protein comprises modules related to the selectin super family and other motifs found in cell surface molecules. Earlier, we demonstrated that SVEP1 is expressed in osteogenic cells both in vivo and in vitro; in the current study we elaborate on the regulation of SVEP1 by 17beta-Estradiol (17betaE2). SVEP1 message is expressed in vivo by bone marrow cells of sham-operated rats, but not in estrogen-depleted ovariectomized (OVX) rats. We demonstrated that 17betaE2 treatment increases the level of the SVEP1 expression in cultured osteoblasts. SVEP1 was identified also in breast carcinoma (BC) cells known to reside in bone when metastasized from the primary tumor. SVEP1 expression was demonstrated by immunohistochemistry and fluorescence-activated cell sorting (FACS) on various BC cell lines. The chromatin immunoprecipitation (ChIP) assay was applied to analyze the estrogen receptor (ER) binding to the putative SVEP1 promoter. We demonstrated that treatment with 17betaE2 or ICI 182,780 affects this binding and regulates the mRNA and protein levels of SVEP1 in BC cells. We propose that SVEP1 may serve as a useful biomarker for studying the mechanism of cells interactions within the local microenvironment affected by estrogen.     

SVEP1 influences monocyte to macrophage differentiation via integrin α4β1/α9β1 and Rho/Rac signalling

BackgroundThe large extracellular matrix protein SVEP1 mediates cell adhesion via integrin α9β1. Recent studies have identified an association between a missense variant in SVEP1 and increased risk of coronary artery disease (CAD) in humans and in mice Svep1 deficiency alters the development of atherosclerotic plaques. However how SVEP1 functionally contributes to CAD pathogenesis is not fully understood. Monocyte recruitment and differentiation to macrophages is a key step in the development of atherosclerosis. Here, we investigated the requirement for SVEP1 in this process.MethodsSVEP1 expression was measured during monocyte–macrophage differentiation in primary monocytes and THP-1 human monocytic cells. SVEP1 knockout THP-1 cell lines and the dual integrin α4β1/α9β1 inhibitor, BOP, were utilised to investigate the effect of these proteins in THP-1 cell adhesion, migration and cell spreading assays. Subsequent activation of downstream integrin signalling intermediaries was quantified by western blotting.ResultsSVEP1 gene expression increases in monocyte to macrophage differentiation in human primary monocytes and THP-1 cells. Using two SVEP1 knockout THP-1 cells we observed reduction in monocyte adhesion, migration, and cell spreading compared to control cells. Similar results were found with integrin α4β1/α9β1 inhibition. We demonstrate reduced activity of Rho and Rac1 in SVEP1 knockout THP-1 cells.ConclusionsSVEP1 regulates monocyte recruitment and differentiation phenotypes through an integrin α4β1/α9β1 dependent mechanism.General significanceThese results describe a novel role for SVEP1 in monocyte behaviour relevant to CAD pathophysiology.     

Oncostatin M regulates mesenchymal cell differentiation and enhances hematopoietic supportive activity of bone marrow stromal cell lines

Bone marrow stromal cell lines (TBR cell lines) established from temperature-sensitive Simian Virus 40 T-antigen gene transgenic mice exhibited myogenic, osteogenic, and adipogenic differentiation. The effect of oncostatin M (OSM) on such mesenchymal cell differentiation of marrow stromal cell lines was examined. One of those stromal cell lines, TBRB, differentiated into skeletal muscle, and its differentiation was stimulated by OSM, whereas differentiation of TBR10-1 into smooth muscle was inhibited by OSM. TBR31-2 is a bipotent progenitor for adipocytes and osteoblasts, and OSM stimulated osteogenic differentiation while inhibiting adipogenic differentiation. On the other hand, TBR cell lines exhibited various potentials for supporting hematopoiesis in culture. When hematopoietic progenitor cells were cocultured with OSM-stimulated stromal cell lines, TBR10-1 and TBR31-2 exhibited enhanced hematopoietic supportive activity. As responsible molecules for stromal cell dependent hematopoiesis, expression of stem cell factor (SCF) (a ligand of c-Kit), vascular cell adhesion molecule (VCAM-1) (a ligand of VLA-4), and secretion of interleukin (IL)-6 were increased by OSM. OSM affected mesenchymal cell differentiation and promoted the hematopoietic supportive activity of marrow stromal cell lines. As OSM production is induced by cytokines from hematopoietic cells, OSM may be a key factor in mutual regulation between hematopoietic cells and stromal cells in the bone marrow. OSM may play a role as a regulator in maintaining the hematopoietic microenvironment in marrow by coordinating mesenchymal differentiation.     

The Casitas B lineage lymphoma (Cbl) mutant G306E enhances osteogenic differentiation in human mesenchymal stromal cells in part by decreased Cbl-mediated platelet-derived growth factor receptor alpha and fibroblast growth factor receptor 2 ubiquitination

Human bone marrow-derived mesenchymal stromal cells (hMSCs) have the capacity to differentiate into several cell types including osteoblasts and are therefore an important cell source for bone tissue regeneration. A crucial issue is to identify mechanisms that trigger hMSC osteoblast differentiation to promote osteogenic potential. Casitas B lineage lymphoma (Cbl) is an E3 ubiquitin ligase that ubiquitinates and targets several molecules for degradation. We hypothesized that attenuation of Cbl-mediated degradation of receptor tyrosine kinases (RTKs) may promote osteogenic differentiation in hMSCs. We show here that specific inhibition of Cbl interaction with RTKs using a Cbl mutant (G306E) promotes expression of osteoblast markers (Runx2, alkaline phosphatase, type 1 collagen, osteocalcin) and increases osteogenic differentiation in clonal bone marrow-derived hMSCs and primary hMSCs. Analysis of molecular mechanisms revealed that the Cbl mutant increased PDGF receptor α and FGF receptor 2 but not EGF receptor expression in hMSCs, resulting in increased ERK1/2 and PI3K signaling. Pharmacological inhibition of FGFR or PDGFR abrogated in vitro osteogenesis induced by the Cbl mutant. The data reveal that specific inhibition of Cbl interaction with RTKs promotes the osteogenic differentiation program in hMSCs in part by decreased Cbl-mediated PDGFRα and FGFR2 ubiquitination, providing a novel mechanistic approach targeting Cbl to promote the osteogenic capacity of hMSCs.     

Supplementation with Fish Oil and Genistein,Individually or in Combination,Protects Bone against the Adverse Effects of Methotrexate Chemotherapy in Rats

Cancer chemotherapy has been shown to induce long-term skeletal side effects such as osteoporosis and fractures; however, there are no preventative treatments. This study investigated the damaging effects of anti-metabolite methotrexate (MTX) subcutaneous injections (0.75 mg/kg BW) for five days and the potential protective benefits of daily oral gavage of fish oil at 0.5 mL/100 g BW (containing 375 mg of n-3 PUFA/100 g BW), genistein (2 mg/100 g BW), or their combination in young adult rats. MTX treatment alone significantly reduced primary spongiosa height and secondary spongiosa trabecular bone volume. Bone marrow stromal cells from the treated rats showed a significant reduction in osteogenic differentiation but an increase in adipogenesis ex vivo. Consistently, stromal cells had significantly higher mRNA levels of adipogenesis-related proliferator activator activated receptor-γ (PPAR-γ) and fatty acid binding protein (FABP4). MTX significantly increased the numbers of bone-resorbing osteoclasts and marrow osteoclast precursor cell pool while significantly enhancing the mRNA expression of receptor activator for nuclear factor kappa B ligand (RANKL), the RANKL/osteoprotegerin (OPG) ratio, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the bone. Supplementary treatment with fish oil and/or genistein significantly preserved trabecular bone volume and osteogenesis but suppressed MTX-induced adipogenesis and increases in osteoclast numbers and pro-osteoclastogenic cytokine expression. Thus, Fish oil and/or genistein supplementation during MTX treatment enabled not only preservation of osteogenic differentiation, osteoblast number and bone volume, but also prevention of MTX treatment-induced increases in bone marrow adiposity, osteoclastogenic cytokine expression and osteoclast formation, and thus bone loss.     

Bone and cartilage formation by skeletal muscle derived cells

In adult individuals when most tissues have progressively lost the ability to regenerate, bone maintains the potential for a continuous self remodeling. The bone marrow has been so far the main recognized source of osteoprogenitor cells that contribute to the turnover of the skeletal scaffold. The possibility though exists that a pool of osteoprogenitor cells resides within other adult tissues and in particular, as reported previously, in other connective tissues such as fat and skeletal muscle. In an attempt to identify an alternative source of osteoprogenitor cells other than bone marrow we looked into the skeletal muscle. A plastic adhering cell population, from now on referred to as skeletal muscle derived cells (SMDCs), was obtained from biopsies of human skeletal muscle. SMDCs were clonogenic and displayed a fibroblast-like morphology. The isolated cell population had a mesenchymal origin as indicated by abundant expression of type I collagen, fibronectin, and vimentin and appeared heterogeneous. SMDCs were positive for alpha smooth actin, and to a lesser extent for desmin and alpha sarcomeric myosin, two specific markers of the myogenic phenotype. Surprisingly though SMDCs expressed early markers of an osteogenic commitment as indicated by positive staining for alkaline phosphatase, osteopontin, and osteonectin. Under the appropriate stimuli, these cells deposited in vitro a mineralized bone matrix and a proteoglycan rich matrix. In addition, SMDCs cultured in the presence of low serum and insulin differentiated towards adipocytes developing abundant lipid droplets in the cytoplasm. Furthermore SMDCs formed three-dimensional bone tissue in vivo when implanted in an immunodeficient mouse, and a mature cartilage rudiment when maintained as a pellet culture. In summary, we report the isolation and characterization of a cell population from the human skeletal muscle not only able to express in vitro specific markers of distinct mesenchymal lineages (adipogenic, chondrogenic, and osteogenic), but most importantly, able to complete the differentiation pathway leading to the formation of bone and cartilage. In this respect SMDCs resemble bone marrow stromal cells (BMSCs).     

Role of Nanog in the maintenance of marrow stromal stem cells during post natal bone regeneration

Post natal bone repair elicits a regenerative mechanism that restores the injured tissue to its pre-injury cellular composition and structure and is believed to recapitulate the embryological processes of bone formation. Prior studies showed that Nanog, a central epigenetic regulator associated with the maintenance of embryonic stem cells (ESC) was transiently expressed during fracture healing, Bais et al. In this study, we show that murine bone marrow stromal cells (MSCs) before they are induced to undergo osteogenic differentiation express ～50× the background levels of Nanog seen in murine embryonic fibroblasts (MEFs) and the W20-17 murine marrow stromal cell line stably expresses Nanog at ～80× the MEF levels. Nanog expression in this cell line was inhibited by BMP7 treatment and Nanog lentivrial shRNA knockdown induced the expression of the terminal osteogenic gene osteocalcin. Lentivrial shRNA knockdown or lentiviral overexpression of Nanog in bone MSCs had inverse effects on proliferation, with knockdown decreasing and overexpression increasing MSC cell proliferation. Surgical marrow ablation of mouse tibia by medullary reaming led to a ～3-fold increase in Nanog that preceded osteogenic differentiation during intramembranous bone formation. Lentiviral shRNA knockdown of Nanog after surgical ablation led to an initial overexpression of osteogenic gene expression with no initial effect on bone formation but during subsequent remodeling of the newly formed bone a ～50% decrease was seen in the expression of terminal osteogenic gene expression and a ～50% loss in trabecular bone mass. This loss of bone mass was accompanied by an increased ～2- to 5-fold adipogenic gene expression and observed increase of fat cells in the marrow space. In summary these data show that Nanog is expressed during surgically induced marrow bone formation and is functionally involved in post natal marrow stromal cell maintenance and differentiation.     

Canonical Wnt signaling is required for Panax notoginseng saponin-mediated attenuation of the RANKL/OPG ratio in bone marrow stromal cells during osteogenic differentiation

Panax notoginseng saponins (PNS) are known to regulate the osteogenic differentiation of bone marrow stromal cells (BMSCs). In the present study, we investigated whether PNS could promote the osteogenic differentiation of BMSCs through modulating the Wnt/β-catenin signaling pathways, which are implicated in BMSCs osteogenesis. We found that PNS enhanced the mRNA expression of OPG, β-catenin, and cyclin D1 while decreased the mRNA expression of RANKL and PPARγ2. The actions of PNS on BMSCs were reversed (or partially) by DKK-1, a classical inhibitor of Wnt/β-catenin signaling. These results suggest that PNS stimulating bone formation by promoting the proliferation and osteogenic differentiation of BMSCs, and could also protect the skeletal system by decreasing bone resorption through reduction of RANKL/OPG expression via Wnt/β-catenin signaling pathways.     

A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture

Two new mAbs (M/K-1 and M/K-2) define an adhesion molecule expressed on stromal cell clones derived from murine bone marrow. The protein is similar in size to a human endothelial cell adhesion molecule known as VCAM-1 or INCAM110. VCAM-1 is expressed on endothelial cells in inflammatory sites and recognized by the integrin VLA-4 expressed on lymphocytes and monocytes. The new stromal cell molecule is a candidate ligand for the VLA-4 expressed on immature B lineage lymphocytes and a possible homologue of human VCAM-1. We now report additional similarities in the distribution, structure, and function of these proteins. The M/K antibodies detected large cells in normal bone marrow, as well as rare cells in other tissues. The antigen was constitutively expressed and functioned as a cell adhesion molecule on cultured murine endothelial cells. It correlated with the presence of mRNA which hybridized to a human VCAM-1 cDNA probe. Partial NH2 terminal amino acid sequencing of the murine protein revealed similarities to VCAM-1 and attachment of human lymphoma cells to murine endothelial cell lines was inhibited by the M/K antibodies. All of these observations suggest that the murine and human cell adhesion proteins may be related. The antibodies selectively interfered with B lymphocyte formation when included in long term bone marrow cultures. Moreover, they caused rapid detachment of lymphocytes from the adherent layer when added to preestablished cultures. The VCAM-like cell adhesion molecule on stromal cells and VLA-4 on lymphocyte precursors may both be important for B lymphocyte formation.     

Molecular cloning and biological activity of a novel Ha-Ras suppressor gene predominantly expressed in skeletal muscle, heart, brain, and bone marrow by differential display using clonal mouse EC cells, ATDC5.

We cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines: embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. The deduced amino acid sequence of A-C1 consists of 167 amino acids and shows 46% identity with that of a ras-responsive gene, rat Ha-rev107. Northern blot analysis showed a distinct hybridization band of 3.2 kilobases. Expression of A-C1 mRNA was detected in undifferentiated ATDC5 cells and myoblastic C2C12 cells, while none of C3H10T1/2 cells, NIH3T3 fibroblasts, Balb/c 3T3 fibroblasts, osteoblastic MC3T3-E1 cells, and ST2 bone marrow stromal cells expressed A-C1 mRNA in vitro. Moreover, A-C1 mRNA was expressed in skeletal muscle, heart, brain, and bone marrow in adult mice. By in situ hybridization, A-C1 gene expression was localized in hippocampus as well as bone marrow cells. By immunocytochemistry, A-C1 protein was detected in the cytoplasm as well as perinuclear region of the cells. Transfection of A-C1 cDNA into Ha-ras-transformed NIH3T3 cell line caused increase in the number of flat colonies and inhibition of cell growth. Our data indicate that A-C1 is expressed in some specific tissues in vivo and modulates Ha-ras-mediated signaling pathway.     

Influence of primary adhesive interactions with fibronectin on clonal growth and osteogenic potential of rat mesenchymal stromal cells

Interactions with the extracellular matrix play an important role in the regulation of cell growth and differentiation. Fibronectin (Fn) is a major component of the extracellular matrix. The impact of Fn and its domains on the adhesion and osteogenic potency of rat mesenchymal stromal cells (MSC) have been evaluated. The examination of bone marrow and fetal liver MSC adhesion dynamics showed that after 7 days of cultivation on Fn the number of adhered clonogenic cells derived from both sources was comparable with their number on plastic but their content in suspension usually was decreased. The population of fetal liver MSC differed from bone marrow-derived MSC in the larger number of cells that adhered for the first 7 days. We found that the primary adhesion of MSC to Fn affected their differentiation potency. Bone marrow MSC cultured on Fn had reduced activity of alkaline phosphatase compared with cells grown on plastic. They deposited a significantly less amount of calcium salts in osteogenic medium. The cultivation of MSC on Fn fragments demonstrated that the crucial role in the inhibition of osteogenesis is played by the Fn cell-binding domain.