Norbert Schütze

Differential regulation of Cbfa1/Runx2 and osteocalcin gene expression by vitamin-D3, dexamethasone, and local growth factors in primary human osteoblasts†

Core binding factor alpha 1 (Cbfa1) is an osteoblast‐specific transcription factor essential to develop a mature osteoblast phenotype. However, its exact role in the signaling of various osteotropic‐differentiating agents is still unclear. In this study, we assessed the effects of 1,25‐(OH)2‐D3 (D3), ascorbic acid, bone morphogenetic protein‐2 (BMP‐2), dexamethasone (Dex), and transforming growth factor‐β (TGF‐β) on Cbfa1 and osteocalcin (OCN) mRNA steady state levels (by semiquantitative RT‐PCR) in an in vitro model of osteoblast differentiation. TGF‐β increased Cbfa1 mRNA levels in normal primary human osteoblasts (pHOB) by 2.6‐fold in a time‐dependent fashion with maximum effect on day 28 (P < 0.001). Similarly, the glucocorticoid Dex enhanced Cbfa1 gene expression by pHOB in a time‐dependent fashion by up to 4.6‐fold (P < 0.001). In contrast, Dex inhibited OCN gene expression levels by 68% (P < 0.01). Treatment with BMP‐2 resulted in an earlier enhancement of Cbfa1 and led to a 4.2‐fold increase with a maximum on day 21 (P < 0.001). Ascorbic acid did not modulate Cbfa1 and OCN gene expression. The effect of vitamin D (D3) on Cbfa1 mRNA expression was influenced by the duration of treatment, being inhibitory after 1 h and having a stimulatory effect after 48 h. Time course experiments indicated a stimulatory effect of D3 on Cbfa1 mRNA levels (by 2.5‐fold after 48 h; P < 0.01). Analysis of the late cellular differentiation marker osteocalcin revealed that D3 increased OCN gene expression by 14‐fold (P < 0.001). In conclusion, in normal primary human osteoblasts, the rapid and pronounced increase of OCN after treatment with D3 seems not to be mediated by Cbfa1. These data imply that Cbfa1 gene expression is differentially regulated by various osteoblastic differentiating agents and is dependent on the stage of maturation. J. Cell. Biochem. 86: 348–356, 2002.

Chondrogenic differentiation of mesenchymal progenitor cells encapsulated in ultrahigh-viscosity alginate.

One major problem of current cartilage repair techniques is that three‐dimensional encapsulated mesenchymal progenitor cells frequently differentiate into hypertrophic cells that express type X collagen and osteogenic marker genes. Studies on wild‐type cells of murine mesenchymal C3H10T1/2 progenitor cells as well as on cells transfected with cDNA encoding for bone morphogenetic protein (BMP)‐2 or ‐4 in alginate revealed that the formation of markers for osteogenesis and chondrogenic hypertrophy apparently depended on the BMP‐transfection. Cells were encapsulated in ultrahigh‐viscosity, clinical grade alginate and differentiation was studied over a period of 17 days. Consistent with results published previously (Biomaterials, 2002;23:2003) staining with haematoxylin–eosin or Alcian blue, immunohistochemical analysis, and quantitative RT‐PCR confirmed the expression of chondrogenic markers (chondroitin‐4‐ and ‐6‐sulfate as well as type II collagen). Production of chondrogenic markers was particularly high in BMP‐4 transfected cells. Hypertrophic chondrogenesis did not occur in BMP‐4 transfected cells, as revealed by measurement of type X collagen, but could be demonstrated for wild‐type cells and to some extent for BMP‐2 transfected cells. The osteogenic markers, type I collagen, alkaline phosphatase, and Cbfal were upregulated in all cell lines even though the levels and the time of upregulation differed significantly. In any case, the markers were less and only very shortly expressed in BMP‐4 transfected cells as revealed quantitatively by real time RT‐PCR. Thus, the in vitro results suggested that BMP‐4 is a very promising candidate for suppressing chondrogenic hypertrophy, while simultaneously enhancing the production of chondrogenic components.

Vitamin D signaling is modulated on multiple levels in health and disease

Vitamin D signaling is dependent on the availability and turnover of the active Vitamin D receptor (VDR) ligand 1,25-dihydroxycholecalciferol and on the efficiency of VDR transactivation. Activating and inactivating secosteroid metabolizing p450 enzymes, e.g. 25-hydroxylases, 1alpha-hydroxylase and 24-hydroxylase, are responsible for ligand availability on the basis of substrate production in the skin and of nutritional intake of precursors. Net availability of active hormone depends on the delivery of substrate and the balance of activating and inactivating enzymes. 1Alpha-hydroxylase is the critical activating enzyme. It is expressed in the kidney for systemic supply and in target tissues for local secosteroid activation. It is upregulated in the kidney by low calcium intake and parathyroid hormone, downregulated by phosphatonins and proinflammatory signal transduction. Transactivation of VDR depends on the correct molecule structure, effective nuclear translocation and the presence of the unliganded heterodimer partner retinoid X-receptor (RXR) and other nuclear cofactors. Rapid Vitamin D-dependent membrane associated effects and consecutive second messenger activation exert an own pattern of gene regulation. A membrane receptor for these effects is hypothesized but not yet identified. Rickets is the long known clinical syndrome of impaired Vitamin D signaling due to Vitamin D3 deficiency. It can be caused by inherited defects of the cascade, nutritional deficits, lack of sunlight exposure, malabsorption and underlying diseases like chronic inflammation. It has been shown during the last decades that many modifiers of Vitamin D signaling are targets of disease in terms of inherited and acquired syndromes and that Vitamin D signaling is modulated at multiple levels and is more complex than mere mechanistic ligand/receptor/DNA interaction.

Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation

BackgroundThe human cysteine rich protein 61 (CYR61, CCN1) as well as the other members of the CCN family of genes play important roles in cellular processes such as proliferation, adhesion, migration and survival. These cellular events are of special importance within the complex cellular interactions ongoing in bone remodeling. Previously, we analyzed the role of CYR61/CCN1 as an extracellular signaling molecule in human osteoblasts. Since mesenchymal stem cells of bone marrow are important progenitors for various differentiation pathways in bone and possess increasing potential for regenerative medicine, here we aimed to analyze the expression of CCN family members in bone marrow-derived human mesenchymal stem cells and along the osteogenic, the adipogenic and the chondrogenic differentiation.ResultsPrimary cultures of human mesenchymal stem cells were obtained from the femoral head of patients undergoing total hip arthroplasty. Differentiation into adipocytes and osteoblasts was done in monolayer culture, differentiation into chondrocytes was induced in high density cell pellet cultures. For either pathway, established differentiation markers and CCN-members were analyzed at the mRNA level by RT-PCR and the CYR61/CCN1 protein was analyzed by immunocytochemistry.RT-PCR and histochemical analysis revealed the appropriate phenotype of differentiated cells (Alizarin-red S, Oil Red O, Alcian blue, alkaline phosphatase; osteocalcin, collagen types I, II, IX, X, cbfa1, PPARγ, aggrecan). Mesenchymal stem cells expressed CYR61/CCN1, CTGF/CCN2, CTGF-L/WISP2/CCN5 and WISP3/CCN6. The CYR61/CCN1 expression decreased markedly during osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. These results were confirmed by immuncytochemical analyses. WISP2/CCN5 RNA expression declined during adipogenic differentiation and WISP3/CCN6 RNA expression was markedly reduced in chondrogenic differentiation.ConclusionThe decrease in CYR61/CCN1 expression during the differentiation pathways of mesenchymal stem cells into osteoblasts, adipocytes and chondrocytes suggests a specific role of CYR61/CCN1 for maintenance of the stem cell phenotype. The differential expression of CTGF/CCN2, WISP2/CCN5, WISP3/CCN6 and mainly CYR61/CCN1 indicates, that these members of the CCN-family might be important regulators for bone marrow-derived mesenchymal stem cells in the regulation of proliferation and initiation of specific differentiation pathways.

Cytokine response of human macrophage-like cells after contact with polyethylene and pure titanium particles

The aim of this study was to establish a human macrophage cell culture system to examine the effect of polyethylene (PE) and titanium particles on cytokine release by macrophage-like cells (MLC) and to quantify this response with respect to the nature and concentration of particles. Human monocytic leukemia cells were differentiated under standard conditions with vitamin D3 and granulocyte macrophage-colony-stimulating factor. Cells were characterized by fluorescence-activated cell-sorter Scan of CD 14 expression analysis as well as a phagocytosis test exploiting fluorescence-labeled particles of bacteria] walls. To achieve a relevant contact between the floating PE particles (approximately 1 microm in size) and MLC, a rotation device was used (15 rotations/min) during incubation. The same was done with the titanium particles. Cell culture supernatants were then analyzed for interleukin (IL)-1beta, IL-8, and tumor necrosis factor (TNF)-alpha using the enzyme-linked immunosorbent assay technique in the absence or presence of particles. Rotation of incubated MLC alone did not influence the secretion of TNF-alpha, but it enhanced secretion of IL-1beta and IL-8 about 30-fold compared to background levels. Both PE and titanium particles significantly enhanced MLC cytokine release, the amount of which depended on the concentration of particles. Using 40 X 10(8) PE particles (0.7 x 10(8) titanium particles) and 10(6) MLC, the maximal release of IL-1beta was about 20-fold (7-fold titanium particles) higher than that of the rotating control sample. The stimulation of IL-8 release was 4-fold (3-fold titanium particles) and of TNF-alpha. 300-fold (170-fold titanium particles) compared to controls. MLC were viable (>90% cell survival) at concentrations less than 108 x 10(8) polyethylene particles per 10(6) MLC and 16 x 10(8) titanium particles per 10(6) MLC. Rotation per se as well as exposure to increasing concentrations of PE and titanium particles stimulates cytokine release (TNF-alpha, IL-1beta, IL-8) by macrophages in vitro. This in vitro model resembles the in vivo situation near arthroplasties, where implant particles make contact with inflammatory cells, such as macrophages. Cytokine release by macrophages may impair osteoblast function as well as stimulate bone resorption by osteoclasts and macrophages, thereby causing aseptic loosening of arthroplasties. Our in vitro model provides a reproducible human cell system that might shed light on the pathogenesis of particle disease and might serve as a reproducible in vitro test system for the biocompatibility of foreign materials.

Formation of cartilage matrix proteins by BMP-transfected murine mesenchymal stem cells encapsulated in a novel class of alginates

Proliferation and differentiation of wild-type, BMP-2 and BMP-4 transfected cells of C3H10T1/2, a mouse mesenchymal stem cell line that can differentiate into chondrocytes, were studied under monolayer (2D-) and encapsulation (3D-) conditions. Cells were encapsulated in a novel class of alginate. The alginate was of clinical grade (CG) because of complete removal of mitogenic and cytotoxic contaminants by chemical means. Compared to commercial alginates used so far for encapsulation it was characterized by ultra-high viscosity (UHV; viscosity of a 0.1% w/v solution of about 20 cP). In contrast to monolayer cultures, proliferation of cells was prevented when the cells were encapsulated in UHV/CG alginate at the same suspension density. As revealed by immunohistochemistry and quantitative RT-PCR, transfected and wild-type monolayer cells showed synthesis of type I collagen after transfer into differentiation medium, while culture in an alginate scaffold resulted in an upregulation of type II collagen and other hyaline cartilage proteins. BMP-4 transfected cells produced considerably more type II collagen than BMP-2 transfected and wild-type cells. BMP-4 transfected cells were also characterized by type I collagen production up to Day 10 and exhibited transient alkaline phosphatase activity levels that were much higher than the peak values observed for the other two cell lines. The coincidence of the ALP peak values with downregulation of type I collagen in BMP-4 transfected cells suggested that C3H10T1/2 cells differentiate into chondrocytes via a chondroprogenitor-like cell.

Plasticity in adipogenesis and osteogenesis of human mesenchymal stem cells.

We established a cell culture system of human mesenchymal stem cells that allows not only for osteogenic and adipogenic differentiation but also for transdifferentiation between both cell lineages. Committed osteoblasts were transdifferentiated into adipocytes with losing osteogenic but highly expressing adipogenic markers. Adipocytes were transdifferentiated into osteoblasts with most of the resulting cells showing osteogenic but some still displaying adipogenic markers apparently not responding to the reprogramming stimulus. Comparing transdifferentiated adipocytes with committed osteoblasts by microarray analysis revealed 258 regulated transcripts, many of them associated with signal transduction, metabolism, and transcription but mostly distinct from established inducing factors of normal adipogenic and osteogenic differentiation, respectively. The regulation pattern of 20 of 22 selected genes was confirmed by semiquantitative RT-PCR. Our results indicate that the plasticity between osteogenesis and adipogenesis extends into the differentiation pathways of both cell lineages and may contribute to the age-related expansion of adipose tissue in human bone marrow.

Differential regulation of blood vessel formation between standard and delayed bone healing

Blood vessel formation is a prerequisite for bone healing. In this study, we tested the hypothesis that a delay in bone healing is associated with an altered regulation of blood vessel formation. A tibial osteotomy was performed in two groups of sheep and stabilized with either a rigid external fixator leading to standard healing or with a highly rotationally unstable one leading to delayed healing. At days 4, 7, 9, 11, 14, 21, and 42 after surgery, total RNA was extracted from the callus. Gene expressions of vWF, an endothelial cell marker, and of several molecules related to blood vessel formation were studied by qPCR. Furthermore, histology was performed on fracture hematoma and callus sections. Histologically, the first blood vessels were detected at day 7 in both groups. mRNA expression levels of vWF, Ang1, Ang2, VEGF, CYR61, FGF2, MMP2, and TIMP1 were distinctly lower in the delayed compared to the standard healing group at several time points. Based on differential expression patterns, days 7 and 21 postoperatively were revealed to be essential time points for vascularization of the ovine fracture callus. This work demonstrates for the first time a differential regulation of blood vessel formation between standard and mechanically induced delayed healing in a sheep osteotomy model.

Catecholestrogens are MCF-7 cell estrogen receptor agonists

Catecholestrogens are important metabolites of estradiol and estrone in the human. Considerable interest has focused on the catecholestrogens 2-hydroxy- and 4-hydroxyestradiol since they bind to the estrogen receptor with an affinity in the range of estradiol. Using the MCF-7 cell line, we analysed the capacity of purified catecholestrogens to transform the estrogen receptor into its high affinity nuclear binding form and to affect receptor-dependent processes such as proliferation and expression of the progesterone receptor (PR). Incubations with 2-hydroxy- and 4-hydroxyestradiol at 10(-8) M for 1 h resulted in tight nuclear binding of the estrogen receptor. During treatment of the cells with catecholestrogens we obtained a marked increase in proliferation rate of 36 and 76% for 2-hydroxy- and 4-hydroxyestradiol, respectively, relative to the inductive effect of estradiol (100%). The PR level, was slightly increased by treatment with 2-hydroxyestradiol (10%), whereas treatment with 4-hydroxyestradiol increased the PR level at 28%, compared to estradiol (100%). From these results we conclude that the 2- and 4-hydroxylated derivatives of estradiol are active hormones and are able to initiate estrogen receptor mediated processes in MCF-7 cells.

The Human Analog of Murine Cystein Rich Protein 16 Is a 1α,25-Dihydroxyvitamin D3 Responsive Immediate Early Gene in Human Fetal Osteoblasts: Regulation by Cytokines, Growth Factors, and Serum1

1α,25-dihydroxyvitamin D3 (1,25-(OH)2D3) is a potent mediator of differentiation and maintenance of specific functions of osteoblasts. To detect novel targets for 1,25-(OH)2D3 action, we applied differential display PCR to human fetal osteoblast-like cells and identified the human analog of murine cystein rich protein 61 (hCYR61) as a 1,25-(OH)2D3-responsive immediate early gene in differentiated fetal osteoblast-like cells. The murine gene CYR61 is important for cell-cell and cell-matrix interactions, and it belongs to an emerging gene family of cysteine-rich proteins. hCYR61 messenger RNA (mRNA) steady-state levels were stimulated 11-fold by 10 nm 1,25-(OH)2D3 by 1 h and declined to control levels by 4 h. This transient stimulation of hCYR61 mRNA was not inhibited by cycloheximide but was prevented by actinomycin D, indicating that the 1,25-(OH)2D3 effect involves transcriptional events and does not require de novo protein synthesis. hCYR61 mRNA stability was not influenced by 1,25(OH)2D3, whereas cyclo...