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ABSTRACT: Transforming growth factor-beta1 (TGF-beta1) action is known to be initiated by its binding to multiple cell surface receptors containing serine/threonine kinase domains that act to stimulate a cascade of signaling events in a variety of cell types. We have previously shown that TGF-beta1 and BMP-2 treatment of primary human osteoblasts (HOBs) enhances cell-substrate adhesion. In this report, we demonstrate that TGF-beta1 elicits a rapid, transient, and oscillatory rise in the intracellular Ca(2+) concentration, [Ca(2+)](i), that is necessary for enhancement of cell adhesion in HOBs but does not alter the phosphorylation state of Smad proteins. This rise in [Ca(2+)](i) in HOB is not observed in the absence of extracellular calcium or when the cells are treated with the L-type Ca(2+) channel blocker, nifedipine, but is stimulated upon treatment with the L-type Ca(2+) channel agonist, Bay K 8644, or under high K(+) conditions. The rise in [Ca(2+)](i) is severely attenuated after treatment of the cells with thapsigargin, a selective endoplasmic reticulum Ca(2+) pump inhibitor. TGF-beta1 enhancement of HOB adhesion to tissue culture polystyrene is also inhibited in cells treated with nifedipine. These data suggest that intracellular Ca(2+) signaling is an important second messenger of the TGF-beta1 signal transduction pathway in osteoblast function.
Journal of Cellular Biochemistry 06/2007; 101(2):348-59. · 2.87 Impact Factor
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ABSTRACT: TGF-β1 is a potent osteoactive factor and exhibits a wide variety of effects on osteoblasts, most of which are mediated through receptor associated Smad proteins. We have recently reported a novel TGF-β1 intracellular Ca2+ signaling pathway in osteoblasts, and found that this signaling is required for the TGF-β1 mediated enhancement of osteoblast adhesion to substrate. Given that interaction between the extracellular matrix protein fibronectin and α5β1 integrin on the cell surface is principally responsible for osteoblast substrate adhesion, we examined here whether the TGF-β1 stimulated Ca2+ signal is involved in this pathway. Our results show that, in primary human osteoblasts, the TGF-β1 induced intracellular Ca2+ signal is responsible, in part, for the stimulation of expression of α5 integrin, but not of β1 integrin or fibronectin. Increased levels of α5 integrin protein and mRNA were seen as early as 12 h after TGF-β1 treatment, but were inhibited by co-treatment of cells with nifedipine, a selective L-type Ca2+ channel blocker. TGF-β1 treatment increased both fibronectin and β1 integrin protein production within 48 h, in a manner unaffected by co-treatment with nifedipine.Immunofluorescence observations revealed that TGF-β1 treatment resulted in increased α5 integrin staining, and more prominent α5 integrin clustering, with increased co-localization with the actin cytoskeleton, effects that were blocked by co-treatment with nifedipine. The TGF-β1 induced intracellular Ca2+ signal in human osteoblasts is thus an important mechanistic step in the regulation of α5 integrin expression, later contributing to enhanced cell adhesion.© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Journal of Orthopaedic Research 12/2005; 20(5):1042 - 1049. · 2.81 Impact Factor
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ABSTRACT: Studies on rodent bone marrow stromal cells (MSCs) have revealed a capacity, for at least a portion of cells, to express neuron-like traits after differentiation in culture. Little, however, is known about the ability of human MSCs in this regard. We show here that incubation with certain differentiation cocktails, particularly those that include reagents that increase cellular cAMP levels, produces a rapid (1-4 h) and transient (24-48 h) transformation of nearly all hMSCs into neuron-like cells displaying a complex network of processes using phase or scanning electron microscopic optics. In addition, differentiated human (h) MSCs express increased quantities of neuron-[beta-tubulin III, neurofilament (NF), neuronal-specific enolase (NSE)] and glial- [glial fibrillary acidic protein (GFAP)] specific proteins and mRNAs, which are also expressed in low levels in undifferentiated MSCs. In contrast, the mesenchymal marker, fibronectin, which is highly expressed in the undifferentiated state, is reduced following differentiation. These biochemical changes, but not the acquisition of a neuron-like appearance, are partially inhibited by incubation of hMSCs with protein (cycloheximide) and mRNA (actinomycin D) synthesis inhibitors with differentiating reagents. Only incubation with 100 ng/ml colchicine, which disrupts the microtubular cytoskeleton, prevents the conversion of hMSCs into neuron- like cells. These results demonstrate that hMSCs acquire the morphological appearance and the biochemical makeup typical of neurons by independently regulated mechanisms.
Stem Cells and Development 01/2005; 13(6):625-35. · 4.46 Impact Factor
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ABSTRACT: We have established a new adult human bone marrow-derived cell line hMPC 32F, stably transduced with human papilloma virus type 16 E6/E7 genes, that displays mesenchymal multilineage differentiation ability in vitro. The hMPC 32F cells exhibited a population doubling time of 22 h and have been maintained in culture for about 20 passages. When cultured in conditions promoting osteogenic, adipogenic, or chondrogenic differentiation, hMPC 32F cells expressed mature differentiated phenotypes. These include (1) osteoblastic phenotype characterized by upregulated alkaline phosphatase (ALP) expression and extracellular matrix mineralization, (2) adipocytic phenotype with the presence of intracellular lipid droplets, and (3) chondrocytic phenotype of round cells surrounded by a sulfated proteoglycan-rich matrix. In addition, the hMPC 32F cells expressed differentiation lineage-specific genes, as detected by RT-PCR. Furthermore, osteogenic and adipogenic cultures responded to regulatory factors such as transforming growth factor-beta1 (TGF-beta1) and 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3). Thus, continuous treatment of osteogenic cultures for 2 weeks with TGF-beta1 decreased ALP activity and mRNA expression and inhibited osteocalcin mRNA expression and matrix mineralization, whereas l,25(OH)2D3 had an additive, stimulatory effect. In adipogenic cultures, treatment with TGF-beta1 for 2 weeks markedly inhibited adipogenesis whereas 1,25(OH)2D3 had no obvious effect. Finally, clonal analysis of hMPC 32F cells revealed a high percentage of multipotent clones, although clones of more restricted differentiation potential were also present. These characteristics of the hMPC 32F cell line suggest their pluripotent, progenitor, and nontransformed nature and indicate their potential application for studying the mechanisms governing developmental potential of adult human bone marrow mesenchymal progenitor cells.
Calcified Tissue International 12/2002; 71(5):447-58. · 2.38 Impact Factor
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ABSTRACT: We have established a new adult human bone marrow-derived cell line hMPC 32F, stably transduced with human papilloma virus type 16 E6/E7 genes, that displays mesenchymal multilineage differentiation ability in vitro. The hMPC 32F cells exhibited a population doubling time of 22 h and have been maintained in culture for about 20 passages. When cultured in conditions promoting osteogenic, adipogenic, or chondrogenic differentiation, hMPC 32F cells expressed mature differentiated phenotypes. These include (1) osteoblastic phenotype characterized by upregulated alkaline phosphatase (ALP) expression and extracellular matrix mineralization, (2) adipocytic phenotype with the presence of intracellular lipid droplets, and (3) chondrocytic phenotype of round cells surrounded by a sulfated proteoglycan-rich matrix. In addition, the hMPC 32F cells expressed differentiation lineage-specific genes, as detected by RT-PCR. Furthermore, osteogenic and adipogenic cultures responded to regulatory factors such as transforming growth factor-b1 (TGF-b1) and 1a, 25-dihydroxyvitamin D3 (1,25(OH)2D3). Thus, continuous treatment of osteogenic cultures for 2 weeks with TGF-b1 decreased ALP activity and mRNA expression and inhibited osteocalcin mRNA expression and matrix mineralization, whereas l,25(OH)2D3 had an additive, stimulatory effect. In adipogenic cultures, treatment with TGF-b1 for 2 weeks markedly inhibited adipogenesis whereas 1,25(OH)2D3 had no obvious effect. Finally, clonal analysis of hMPC 32F cells revealed a high percentage of multipotent clones, although clones of more restricted differentiation potential were also present. These characteristics of the hMPC 32F cell line suggest their pluripotent, progenitor, and nontransformed nature and indicate their potential application for studying the mechanisms governing developmental potential of adult human bone marrow mesenchymal progenitor cells.
Calcified Tissue International 10/2002; 71(5):447-458. · 2.38 Impact Factor
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ABSTRACT: TGF-beta1 is a potent osteoactive factor and exhibits a wide variety of effects on osteoblasts, most of which are mediated through receptor associated Smad proteins. We have recently reported a novel TGF-beta1 intracellular Ca2+ signaling pathway in osteoblasts, and found that this signaling is required for the TGF-beta1 mediated enhancement of osteoblast adhesion to substrate. Given that interaction between the extracellular matrix protein fibronectin and alpha5beta1 integrin on the cell surface is principally responsible for osteoblast substrate adhesion, we examined here whether the TGF-beta1 stimulated Ca2+ signal is involved in this pathway. Our results show that, in primary human osteoblasts, the TGF-beta1 induced intracellular Ca2+ signal is responsible, in part, for the stimulation of expression of alpha5 integrin, but not of beta1 integrin or fibronectin. Increased levels of alpha5 integrin protein and mRNA were seen as early as 12 h after TGF-beta1 treatment, but were inhibited by co-treatment of cells with nifedipine, a selective L-type Ca2+ channel blocker. TGF-beta1 treatment increased both fibronectin and beta1 integrin protein production within 48 h, in a manner unaffected by co-treatment with nifedipine. Immunofluorescence observations revealed that TGF-beta1 treatment resulted in increased alpha5 integrin staining, and more prominent alpha5 integrin clustering, with increased co-localization with the actin cytoskeleton, effects that were blocked by co-treatment with nifedipine. The TGF-beta1 induced intracellular Ca2+ signal in human osteoblasts is thus an important mechanistic step in the regulation of alpha5 integrin expression, later contributing to enhanced cell adhesion.
Journal of Orthopaedic Research 10/2002; 20(5):1042-9. · 2.81 Impact Factor
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ABSTRACT: Marrow stroma-derived cells (MSC) are highly proliferative, multipotential cells that have been considered as ideal candidate cells for autologous tissue engineering applications. In this study, we have characterized the chondrogenic potential of human MSCs in both a PLA/alginate amalgam and pure PLA macrostructure as model three-dimensional constructs to support both chondrogenic differentiation and proliferation following TGF-beta treatment. MSCs were seeded in experimental groups that consisted of PLA-loaded constructs and PLA/alginate amalgams with and without recombinant human TGF-beta1. Chondrogenesis of the PLA and the PLA/alginate amalgam cultures was assessed at weekly intervals by histology, immunohistochemistry, scanning electron microscopy, sulfate incorporation, and RT-PCR. Chondrogenic differentiation occurs within a polymeric macrostructure with TGF-beta1 treatment as indicated by histological, immunohistochemical, sulfate incorporation, and gene expression profiles. This macrostructure can be further encased in an alginate gel/solution to optimize cell shape and to confine growth factors and cells within the polymer construct, while the polymeric scaffold provides appropriate mechanical/tissue support. The stable three-dimensional PLA/alginate amalgam represents a novel candidate system of mesenchymal chondrogenesis, which is amendable to investigation of mechanical and biological factors that normally modulate cartilage development and formation as well as a potential tissue engineering construct for cartilage repair.
Annals of the New York Academy of Sciences 07/2002; 961:134-8. · 3.15 Impact Factor
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ABSTRACT: The osteoactive factor, transforming growth factor beta1 (TGF-beta1), influences osteoblast activity and bone function. We recently characterized a Smad-independent TGF-beta1-induced Ca(2+) signal in human osteoblasts (HOB) and demonstrated its importance in cell adhesion. Here, we further elucidate the role of the TGF-beta1 Ca(2+) signal in the mechanics of HOB adhesion. Osteoblast interaction with fibronectin (FN) through alpha5beta1 integrin is principally responsible for osteoblast-substrate adhesion. Our results show that the TGF-beta1 intracellular Ca(2+) signal is responsible, in part, for stimulation of alpha5 integrin expression, but not beta1 integrin or FN expression. Increased alpha5 integrin protein and mRNA expression was seen as early as 12 h after TGF-beta1 treatment, but was inhibited by cotreatment with nifedipine, a Ca(2+) channel blocker. TGF-beta1 increased both FN and beta1 integrin protein production within 48 h, independent of nifedipine cotreatment. Immunofluorescence observations revealed that TGF-beta1 increased alpha5 integrin staining, clustering, and colocalization with the actin cytoskeleton, effects that were blocked by nifedipine. The TGF-beta1 Ca(2+) signal, a pathway crucial for HOB adhesion, enhances alpha5 integrin expression, focal contact formation, and cytoskeleton reorganization. These early events are necessary for osteoblast adhesion; thus they determine the fate of the cell and ultimately affect bone function.
Annals of the New York Academy of Sciences 07/2002; 961:178-82. · 3.15 Impact Factor
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ABSTRACT: Bone marrow-derived cells are considered as candidate cells for cartilage tissue engineering by virtue of their ability to undergo chondrogenesis in vitro when cultured in high density or when embedded within a three-dimensional matrix in the presence of growth factors. This study evaluated the potential of human bone marrow-derived cells for cartilage tissue engineering by examining their chondrogenic properties within a three-dimensional amalgam scaffold consisting of the biodegradable polymer, poly-L-lactic acid (PLA) alone, and with the polysaccharide gel, alginate. Cells were suspended either in alginate or medium and loaded into porous PLA blocks. Alginate was used to improve cell loading and retention within the construct, whereas the PLA polymeric scaffold provided appropriate mechanical support and stability to the composite culture. Cells seeded in the PLA/alginate amalgams and the plain PLA constructs were treated with different concentrations of recombinant human transforming growth factor-beta1 (TGF-beta 1) either continuously (10 ng/mL) or only for the initial 3 days of culture (50 ng/mL). Chondrogenesis was assessed at weekly intervals with cultures maintained for up to 3 weeks. Histological and immunohistochemical analysis of the TGF-beta 1-treated PLA/alginate amalgam and PLA constructs showed development of a cartilaginous phenotype from day 7 to day 21 as demonstrated by colocalization of Alcian blue staining with collagen type II and cartilage proteoglycan link protein. Expression of cartilage specific genes, including collagen types II and IX, and aggrecan, was detected in TGF-beta 1-treated cultures by reverse transcription-polymerase chain reaction analysis. The initiation and progression of chondrogenic differentiation within the polymeric macrostructure occurred with both continuous and the initial 3-day TGF-beta 1 treatment regimens, suggesting that key regulatory events of chondrogenesis take place during the early period of cell growth and proliferation. Scanning electron microscopy revealed abundant cells with a rounded morphology in the PLA/alginate amalgam. These findings suggest that the three-dimensional PLA/alginate amalgam is a potential candidate bioactive scaffold for cartilage tissue engineering applications.
Journal of Biomedical Materials Research 01/2002; 57(3):394-403.
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ABSTRACT: Mesenchymal stem cells are a rare population of undifferentiated cells, isolated from adult tissue sources, that have the capacity to differentiate into mesodermal lineages, including bone, fat, muscle, cartilage, tendon, and marrow stroma. These cell populations may be expanded in culture and subsequently permitted to differentiate into the desired lineage. This directed differentiation may be reached by the application of bioactive molecules, specific growth factors, and signaling molecules. Understanding the functional potential of these cells and the signaling mechanisms underlying their differentiation should lead to innovative protocols for clinical orthopaedic interventions. Clinically applicable techniques to isolate, expand, and reimplant these autogenous cells will become part of the repertoire of orthopaedic therapy. In the presence of extrinsic signaling molecules, provided by both the clinician and the local cellular environment, the intrinsic multipotential nature of the stem cells may be realized for applications such as the replacement of bone graft for segmental defects, nonunions, and spinal fusions. Additional applications may include treatment of full-thickness articular defects and articular resurfacing by site-specific delivery of stem cells. The ultimate goal is directed cellular regeneration of damaged or diseased musculoskeletal tissue. Currently, the limitation is our knowledge and ability to direct this differentiation, but with further study molecular orthopaedic interventions should become a reality.
MedGenMed: Medscape general medicine 03/2001;
