Colony stimulating factor-1 stimulates Ishikawa cell proliferation and lipocortin II synthesis.

Proliferation of the Ishikawa human endometrial adenocarcinoma cell line is under the concerted control of oestrogen and progesterone. Here we demonstrate that Ishikawa cells express colony stimulating factor-1 (CSF-1), CSF-1 receptor mRNA and are growth stimulated by CSF-1 treatment. An early event associated with CSF-1 treatment is the induction of lipocortin II synthesis, a protein whose expression is also under oestrogen and progesterone control. However, neither CSF-1 or CSF-1 receptor mRNA appear to be modulated by oestrogen or progesterone.     

The expression of the fibrillar collagen genes during fracture healing: heterogeneity of the matrices and differentiation of the osteoprogenitor cells

The cells that express the genes for the fibrillar collagens, types I, II, III and V, during callus development in rabbit tibial fractures healing under stable and unstable mechanical conditions were localized. The fibroblast-like cells in the initial fibrous matrix express types I, III and V collagen mRNAs. Osteoblasts, and osteocytes in the newly formed membranous bone under the periosteum, express the mRNAs for types I, III and V collagens, but osteocytes in the mature trabeculae express none of these mRNAs. Cartilage formation starts at 7 days in calluses forming under unstable mechanical conditions. The differentiating chondrocytes express both types I and II collagen mRNAs, but later they cease expression of type I collagen mRNA. Both types I and II collagens were located in the cartilaginous areas. The hypertrophic chondrocytes express neither type I, nor type II, collagen mRNA. Osteocalcin protein was located in the bone and in some cartilaginous regions. At 21 days, irrespective of the mechanical conditions, the callus consists of a layer of bone; only a few osteoblasts lining the cavities now express type I collagen mRNA.We suggest that osteoprogenitor cells in the periosteal tissue can differentiate into either osteoblasts or chondrocytes and that some cells may exhibit an intermediate phenotype between osteoblasts and chondrocytes for a short period. The finding that hypertrophic chondrocytes do not express type I collagen mRNA suggests that they do not transdifferentiate into osteoblasts during endochondral ossification in fracture callus.     

Mechanical stretch-induced changes in cell morphology and mRNA expression of tendon/ligament-associated genes in rat bone-marrow mesenchymal stem cells

It has been demonstrated that mechanical stimulation plays a vital role in regulating the proliferation and differentiation of stem cells. However, little is known about the effects of mechanical stress on tendon/ligament development from mesenchymal stem cells (MSCs). Here, using a custom-made cell-stretching device, we studied the effects of mechanical stretching on the cell morphology and mRNA expression of several key genes modulating tendon/ligament genesis. We demonstrate that bone-marrow-derived rat MSCs (rMSCs), when subjected to cyclic uniaxial stretching, express obvious detectable mRNAs for tenascin C and scleraxis, a unique maker of tendon/ligament formation, and significantly increased levels of type I collagen and type III collagen mRNAs. The stretched cells also orient at approximately 65 degrees with respect to the stretching direction and exhibit a more fibroblast-like morphology. Collectively, these results indicate that mechanical stretching facilitates the directed differentiation of rMSCs into tendon/ligament fibroblasts, which has potential implications for the tissue engineering of bioartificial tendons and ligaments.     

Retinoic acid effects on an SV-40 large T antigen immortalized adult rat bone cell line

Clonal cell lines were established from adult rat tibia cells immortalized with SV-40 large T antigen. One clone (TRAB-11), in which retinoic acid (RA) induced alkaline phosphatase (AP) activity, was selected for further study. The TRAB-11 cells express high levels of type I collagen mRNA, type IV collagen, fibronectin, practically no type III collagen, little osteopontin, and no osteocalcin. RA stimulates proliferation of TRAB-11 cells (starting at 10 pM) and survival (starting at 100 pM). TRAB-11 cells synthesize fibroblast growth factor-2 (FGF-2), which has potent autocrine mitogenic effects on these cells and acts synergistically with RA. TRAB-11 cells attach better to type IV collagen than to fibronectin or laminin. Cell attachment to type IV collagen is increased by RA and decreased (65%) by an antibody directed against alpha1beta1 integrin. RA up-regulates steady-state levels of alpha1, mRNA without affecting beta1 mRNA expression. In conclusion, we report the establishment of a clonal cell line from the outgrowth of adult rat tibiae which is highly sensitive to RA in its growth and survival in culture, apparently as a result of integrin-mediated cell interaction with extracellular matrix proteins.     

Cultured mouse marrow stromal cell lines. II. Distinct subtypes differing in morphology, collagen types, myelopoietic factors, and leukemic cell growth modulating activities

A series of stromal cell lines were studied for their growth properties, electron microscopic morphology, cytochemical profile, collagen types, production of myelopoietic factors, and modulation of leukemic cell growth. Three cell types were identified in addition to the previously described macrophages (14M and 14M1) and preadipocytes (14F). MBA-1 cells were found to be fibroblasts by their ability to synthesize collagen types I and III, while the cell line MBA-13 shared properties in common with both fibroblasts and endothelial cells (collagen types I, III, IV, V). The third cell type, represented by the stromal cell line MBA-2, produced mainly collagen types IV and V and exhibited junctional complexes between adjacent cells. All of the cell lines tested produced and secreted a macrophage-colony-stimulating factor, CSF-1. MBA-2 and to a lesser extent, MBA-13, produced an additional activity resistant to anti-CSF-1 antiserum. Trypsin extraction of outer surface components from two clones of the MBA-2 cell line (MBA-2.1 and MBA-2.4) yielded high molecular weight factor(s) that specifically inhibited the growth of a plasmacytoma cell line (MPC-11). Such inhibitory activity was not detected in other stromal cell lines. It is possible that this variability in the nature of stromal cell lines represents corresponding diversity of cell types comprising the hematopoietic microenvironment in vivo.     

Changes in the content of the fibrillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing

Summary The menisci are first seen as triangular aggregations of cells in the 20-day rabbit fetus. At 25-days, a matrix that contains types I, III and V collagens has formed. These collagens are also found in the 1-week neonatal meniscus, but by 3 weeks, type II collagen is present in some regions. By 12 to 14 weeks, typically cartilaginous areas with large cells in lacunae are found and by 2 years, these occupy the central regions of the inner two-thirds of the meniscus. The surface layers of the meniscus contain predominantly type I collagen. From 12 to 14 weeks onwards, there is little overlap between the regions with types I or II collagens, that is, these are discrete regions of type I-containing fibrocartilage and type II-containing cartilage. Types III and V collagens are found throughout the menisci, particularly in the pericellular regions. All the cells in the fetal and early neonatal menisci express the mRNA for type I collagen. At 3 weeks postnatal, cells that express type I collagen mRNA are found throughout the meniscus, but type II collagen mRNA is expressed only in the regions of developing cartilage. At 12- to 14-weeks, only type II collagen mRNA is expressed, except at the periphery next to the ligament where a few cells still express type I collagen mRNA. Rabbit menisci, therefore, undergo profound changes in their content and arrangement of collagens during postnatal development.     

Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts

A mouse genomic clone was isolated by cross-hybridization with a DNA fragment which codes for the NH2-propeptide of chick alpha1(III) collagen. The region of cross-hybridization within the mouse clone was localized, its sequence determined, and an exon coding for the NH2-propeptide of mouse alpha1(III) collagen was identified. This DNA fragment hybridizes to an RNA species of approximately 5300 nucleotides, slightly larger than the major alpha2(I) collagen RNA species. The mouse type III collagen probe was used to examine the effect of transformation on alpha1(III) collagen RNA levels in mouse fibroblasts. The levels of type III and type I collagen mRNA levels were compared in control and sarcoma virus-transformed murine cell lines, as well as in NIH 3T3 cells transformed by members of the human ras oncogenes. The levels of type III RNA decreased about 10-15-fold in Moloney sarcoma virus-transformed cells and in a cell line transformed with a v-mos-containing plasmid, but showed only a 50% decrease in a Kirsten murine sarcoma virus-transformed BALB 3T3 cell line, and increased 4-fold in a Rous sarcoma virus (RSV)-transformed BALB 3T3 cell line. In contrast, the levels of alpha2(I) collagen mRNA are 8- to 10-fold lower in all these cell lines when compared to untransformed cells. NIH 3T3 cells transformed with two human ras oncogenes showed decreased levels of alpha2(I) and alpha1(III) mRNAs. In contrast to the RSV-transformed mouse cell line, RSV-transformed chick embryo fibroblasts contained much smaller amounts of type III RNA than control chick embryo fibroblasts. We conclude that the levels of alpha1(III) and alpha2(I) collagen mRNA are often but not necessarily coordinately regulated by transformation in mouse cells.     

Gene expression of type I and type III collagen by mechanical stretch in anterior cruciate ligament cells

Mechanical stretch affects the healing and remodeling process of the anterior cruciate ligament (ACL) after surgery in important ways. In this study, the effects of mechanical stress on gene expression of type I and III collagen by cultured human ACL cells and roles of transforming growth factor (TGF)-beta1 in the regulation of mechanical strain-induced gene expression were investigated. Uniaxial cyclic stretch was applied on ACL cells at 10 cycles/min with 10% length stretch for 24 h. mRNA expression of the type I and type III collagen was increased by the cyclic stretch. TGF-beta1 protein in the cell culture supernatant was also increased by the stretch. In the presence of anti-TGF-beta1 antibody, stretch-induced increase in type I and type III mRNA expression was markedly ablated. The results suggest that the stretch-induced mRNA expression of the type I and type III collagen is mediated via an autocrine mechanism of TGF-beta1 released from ligament cells.     

Involvement of CSF-1 in generating a stroma-independent hematopoietic stem cell line

The hematopoietic stem cell line, Myl-D7, is maintained by a self-renewing stem cell population that spontaneously generates myeloid, lymphoid, and erythroid progeny. MS-5 stromal cells are necessary for the growth of Myl-D7 cells. One component of the Myl-D7 cells proliferation activity released by MS-5 stromal cells was enriched by Q sepharose fractionation and shown to be colony stimulating factor-1 (CSF-1) by Western blotting, BAC1.2F5 cell bioassay and inhibition of Myl-D7 proliferation by CSF-1 antibody. The requirement of Myl-D7 cells for CSF-1 was also demonstrated independently by selecting for rare, stroma-independent Myl-D7 mutant clones able to grow without stroma and additional factors. Eighty-nine stroma-independent mutant clones were obtained and belonged to two classes. The majority of mutants did not secrete any growth promoting activity. The second, rarer class of mutants releases a factor that stimulates proliferation/survival for up to several months and approximately half of the secretors express high levels of CSF-1 mRNA. Wild type Myl-D7 grown with supernatants from the secretor cells retained the stem cell phenotype. These data suggest that CSF-1 may act as a key factor in stroma-regulated hematopoiesis and cell-cell interaction.     

Persistent Activation of Mitogen-Activated Protein Kinases p42 and p44 and ets-2 Phosphorylation in Response to Colony-Stimulating Factor 1/c-fms Signaling

An antibody that specifically recognized phosphothreonine 72 in ets-2 was used to determine the phosphorylation status of endogenous ets-2 in response to colony-stimulating factor 1 (CSF-1)/c-fms signaling. Phosphorylation of ets-2 was detected in primary macrophages, cells that normally express c-fms, and in fibroblasts engineered to express human c-fms. In the former cells, ets-2 was a CSF-1 immediate-early response gene, and phosphorylated ets-2 was detected after 2 to 4 h, coincident with expression of ets-2 protein. In fibroblasts, ets-2 was constitutively expressed and rapidly became phosphorylated in response to CSF-1. In both cell systems, ets-2 phosphorylation was persistent, with maximal phosphorylation detected 8 to 24 h after CSF-1 stimulation, and was correlated with activation of the CSF-1 target urokinase plasminogen activator (uPA) gene. Kinase assays that used recombinant ets-2 protein as a substrate demonstrated that mitogen-activated protein (MAP) kinases p42 and p44 were constitutively activated in both cell types in response to CSF-1. Immune depletion experiments and the use of the MAP kinase kinase inhibitor PD98059 indicate that these two MAP kinases are the major ets-2 kinases activated in response to CSF-1/c-fms signaling. In the macrophage cell line RAW264, conditional expression of raf kinase induced ets-2 expression and phosphorylation, as well as uPA mRNA expression. Transient assays mapped ets/AP-1 response elements as critical for basal and CSF-1-stimulated uPA reporter gene activity. These results indicate that persistent activation of the raf/MAP kinase pathway by CSF-1 is necessary for both ets-2 expression and posttranslational activation in macrophages.     

Type III collagen is essential for growth acceleration of human osteoblastic cells by ascorbic acid 2-phosphate, a long-acting vitamin C derivative.

Collagen has been reported to be essential for the proliferation of various kinds of cells including human osteoblastic cells [Takamizawa, S., Maehata, Y., Imai, K., Senoo, H., Sato, S., Hata, R., 2004. Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells. Cell Biol. Int. 28, 255-265], but the type(s) of collagen responsible for growth regulation is not known. Presently we found that ascorbic acid 2-phosphate, a long-acting vitamin C derivative, stimulated both cell growth and the expression of mRNA for type III collagen in human osteoblast-like MG-63 cells and in normal human osteoblasts, as well as in human bone marrow mesenchymal stem cells, but not the expression of type I collagen in these cells. Epidermal growth factor also stimulated both cell growth and expression of type III collagen mRNA in MG-63 cells. Among MG-63 cell clones, their growth rates correlated significantly with their COL3A1 messenger RNA levels but not with their COL1A1 or COL1A2 messenger RNA levels. Transfection of MG-63 cells with siRNA for COL3A1 but not with that for COL1A1 decreased the growth rates of the transfected cells concomitant with a drop in the level of COL3A1 mRNA. Furthermore, cell proliferation as observed by thymidine incorporation into DNA and cell number was increased when MG-63 cells were cultured on type III collagen-coated dishes. Taken together, our results indicate that type III collagen is the collagen component responsible for the growth stimulation of human osteoblastic cells.     

Osteoblastic gene expression during adipogenesis in hematopoietic supporting murine bone marrow stromal cells

A growing body of data suggests that the bone marrow stroma contains a population of pluripotent cells capable of differentiating into adipocytes, osteoblasts, and lymphohematopoietic supporting cells. In this work, the murine stromal cell lines BMS2 and +/+ 2.4 have been examined as preadipocytes and adipocytes for evidence of osteoblastic gene expression. Adipocyte differentiation has been quantitated using fluorescence activated cell sorting. Within 7–10 days of adipocyte induction by treatment with glucocorticoids, indomethacin, and methylisobutylxanthine, between 40% to 50% of the cells contain lipid vacuoles and exhibit a characteristic adipocyte morphology. Based on immunocytochemistry, both the adipocytes and preadipocytes express a number of osteoblastic markers; these include alkaline phosphatase, osteopontin, collagen (I, III), bone sialoprotein II, and fibronectin. Based on biochemical assays, the level of alkaline phosphatase expression is not significantly different between preadipocyte and adipocyte cells. However, unlike rat cell lines, dexamethasone exposure causes a dose-dependent decrease in enzyme activity. The steady-state mRNA levels of the osteoblast associated genes varies during the process of adiopogenesis. The relative level of collagen I and collagen III mRNA is lower in adipocyte-induced cells when compared to the uninduced controls. Osteocalcin mRNA is detected in preadipocytes but absent in adipocytes. These data indicate that osteoblastic gene expression is detected in cells capable of undergoing adipocyte differentiation, consistent with the hypothesis that these cell lineages are interrelated. © 1993 Wiley-Liss, Inc.     

Defective post-translational modification of collagen IV in a mutant F9 teratocarcinoma cell line is associated with delayed differentiation and growth arrest in response to retinoic acid

We have selected a mutant F9 teratocarcinoma stem cell line, RA-5-1, which does not exhibit normal differentiation into parietal endoderm in the presence of retinoic acid, dibutyryl cyclic AMP, and theophylline (RACT). In this report, we demonstrate that the RA-5-1 mutant possesses a prolyl-4-hydroxylase enzyme with a higher Km for a synthetic collagen substrate and that this alteration results in a 6-7-fold reduction in the amount of collagen IV in the medium of RACT-treated mutant cells, as compared to wild type F9 cells. In addition, the collagen IV that is secreted by RACT-treated RA-5-1 cells has an abnormally low molecular weight and contains 6-9-fold less 4-hydroxyproline than the collagen IV secreted by RACT-treated wild type F9 cells. A brief ascorbate treatment can increase the hydroxyproline content of the collagen IV secreted by RACT-treated RA-5-1 cells. A large reduction in the amount of laminin in the medium of RACT-treated RA-5-1 mutant cells is also observed. Concomitant with the reduction in collagen IV and laminin polypeptides in the medium, the expression of several other differentiation-specific mRNAs is delayed in the RACT-treated RA-5-1 cells relative to wild type F9 cells. Moreover, the mutant cells do not exhibit the morphology or the complete growth arrest of wild type terminally differentiated parietal endoderm cells in the presence of RACT. These results suggest that a defect in the post-translational modification of collagen IV in the mutant RA-5-1 prevents the complete expression of the differentiation program in response to RACT. These experiments also demonstrate that the expression of certain differentiation-specific genes is compatible with continued proliferation in the mutant line.     

Changes in the distribution of fibrillar collagens in the collateral and cruciate ligaments of the rabbit knee joint during fetal and postnatal development

Summary The collateral ligaments can be clearly distinguished in the 25-day fetal rabbit knee joint. Types I and V collagens are present in the extracellular matrix between the cells of the lateral and medial collateral ligaments and this distribution persists until the rabbit is skeletally mature. From 8 months onwards type III collagen is also present, particularly around the cells. Type I collagen mRNA is expressed by the cells from the 25-day fetal to 8-month-old adult ligament. The ligament sheath is composed of types III and V collagens. The cruciate ligaments are present between the femur and tibia in the 20-day fetus. The matrix is composed of types I and V collagens from the 25-day fetus until at 12- to 14-weeks postnatal, type III collagen appears in the pericellular regions together with type V. At 8 months and 2 years, the amount of type III collagen has increased. All the cells express the mRNA for type I collagen at 12- to 14-weeks, but only isolated cells express this mRNA at 8 months. Thus, both the collateral and cruciate ligaments undergo changes in their complement of collagens during postnatal development and ageing. The implications of these complex interactions of different types of collagen are discussed in relation to healing and the surgical replacement of torn ligaments by tendons.     

A colony-stimulating factor 1 (CSF-1) receptor/platelet-derived growth factor-beta receptor gene fusion confers CSF-1 independence and tumorigenicity on a c-myc-immortalized monocyte cell line.

Monocytes and macrophages express the receptor for the hematopoietic growth factor colony-stimulating factor 1 (CSF-1) and require this factor for growth in culture. A murine monocyte tumor cell line that lacks the usual requirement for CSF-1 was isolated. On the basis of the similarity of the structures of the CSF-1 and platelet-derived growth factor (PDGF) receptors and because monocytes normally secrete PDGF, we analyzed the tumor cell line for anomalous expression of the PDGF-R beta gene. Two different cDNAs that each contain sequences corresponding to the complete coding sequence of PDGF-R beta fused (in frame) to the amino-terminal half of the CSF-1 receptor were isolated. Introduction of these PDGF-R beta-related cDNAs into two partially transformed, CSF-1-dependent monocyte cell lines resulted in autonomous growth and cell transformation. These monocyte cell lines exhibit a novel form of growth factor receptor activation that can lead to oncogenic growth in collaboration with the c-myc oncogene.     

Mouse mesangial cells produce colony-stimulating factor-1 (CSF-1) and express the CSF-1 receptor

CSF-1 stimulates the survival, proliferation, and differentiation of mononuclear phagocytes and may also play a role in placental development. The expression of CSF-1 and the CSF-1 receptor (CSF-1R) and their regulation were examined in cultures of mouse mesangial cells (MC). The concentration of CSF-1 in the medium of cultured MC increased linearly with time over 24 h. IFN-gamma stimulated and dibutyryl cyclic AMP inhibited CSF-1 production in a dose-dependent manner. MC expression of CSF-1 mRNA was shown by Northern blot analysis, and CSF-1 mRNA levels were increased within 4 h of IFN-gamma addition and inhibited within 4 h of dibutyryl cyclic AMP addition. Indirect immunofluorescence indicated that 90% of the untreated cultured MC expressed CSF-1. In addition, CSF-1R expression by MC was demonstrated by immunofluorescence with anti-receptor antibody, specific binding of [125I] CSF-1, and expression of the CSF-1R mRNA by Northern blot analysis. Thus, mouse MC, specialized pericytes of non-bone marrow origin, not only produce CSF-1 but also express receptors for CSF-1. The effects of CSF-1 on MC may be important in the control of immune function in the glomerulus.