Differentiation and Cell Surface Expression of Transforming Growth Factor-β Receptors Are Regulated by Interaction with Matrix Collagen in Murine Osteoblastic Cells

Abstract

Although transforming growth factor (TGF)-␤ enhances bone formation, it inhibits the differentiation of osteoblasts. To clarify the regulatory mechanism of osteoblastic differentiation and TGF-␤ actions, the relationship among differentiation, TGF-␤ actions, and matrix protein synthesis was examined using murine osteoblast-like MC3T3-E1 cells. Alkaline phosphatase (ALP) activity continued to increase during long-term cultures, and the increase was closely associated with a reduction in cell surface TGF-␤ receptors competent to bind TGF-␤. Both the stimulation of proteoglycan synthesis and the inhibition of ALP activity by TGF-␤ were also suppressed. Collagen synthesis inhibitors and an anti-␣2␤1 integrin blocking antibody blocked the changes in ALP activity and TGF-␤ receptors, and a DGEA peptide that interferes binding of collagen to ␣2␤1 integrin also blocked the increase in ALP activity. Furthermore, when MC3T3-E1 cells were cultured on extracellular matrix layers obtained from these cells, all the differentiation-associated changes could be observed without collagen production, and the extracellular matrix-induced differentiation was also blocked by an anti-␣2␤1 integrin antibody. These results demonstrate that the interaction of cell surface ␣2␤1 integrin with matrix collagen synthesized by osteoblasts themselves is involved in the osteoblastic differentiation and the reduction in cell surface receptors and actions of TGF-␤. It is suggested that matrix collagen synthesized under the stimulation by TGF-␤ plays an important role in the regulation of osteoblastic differentiation and TGF-␤ actions by differentiation-associated down-regulation of TGF-␤ receptors.Cells of osteoblast lineage exert various functions to maintain bone formation. After bone resorption, osteoblast precursors migrate and proliferate at the site of bone formation. They, then, synthesize type I collagen and other matrix proteins. Onto the newly formed unmineralized matrices, hydroxyapatite crystals are accumulated, and mineralization of bone is completed (1). In order to form lamellar bones that maintain structural integrity and physical strength, it appears to be of critical importance for osteoblastic cells to maintain sequentially ordered development of these multiple functions.Using cultured osteoblastic cells obtained from bone or of clonal origin, it has become clear that these cells express various functional properties in a differentiation-dependent manner from the early proliferation phase, via the matrix formation phase with active matrix protein synthesis, to the mineralization phase (1-3). Various hormones and cytokines are shown to affect the differentiation process and functional properties of these cells. However, the mechanism that controls the differentiation of osteoblastic cells is as yet unclear.Transforming growth factor-␤ (TGF-␤) 1 is stored in bone matrix as a latent form (4), is thought to be released and activated during osteoclastic bone resorption (5), and to play an important role in the regulation of bone meta...

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

“…Our results suggest that FAK, ERK, Runx2/Cbfa1, and osterix constitute important elements of the signal transduction machinery controlling this process. Considerable work has focused on the role of integrin-mediated signaling during progression of osteoblast differentiation [15,17,18,32,33], but our work focuses on a much earlier step, when hMSC are still capable of differentiating into multiple cell types. Clearly, a different set of decisions are being made by hMSC at this earlier step of osteogenesis, in that down-regulation of adipogenic, chondrogenic, neural, and muscular genes is occurring at the same time that osteogenic genes are being upregulated [34,35].…”

Differential Morphology and Homogeneity of Tissue-Engineered Cartilage in Hydrodynamic Cultivation with Transient Exposure to Insulin-Like Growth Factor-1 and Transforming Growth Factor-β1

“…It is believed that overdoses of growth factors can suppress expression of their own receptors 34,35 possibly through enhanced receptor degradation. 36 Futureinquiries into changes in receptor numbers due to their interactions with other environmental factors such as ECM 37 are necessary. Besides, rapid IGF-1 accumulation in culture media (Fig.…”

Morphological influence of ascorbic acid deficiency on endochondral ossification in osteogenic disorder Shionogi rat

“…Consequently, decreased bone volume appears to have been caused primarily by the reduced number of active osteoblasts. Because the production of type I collagen is one of the earliest events induced by AsA in cultured osteoblastic cells, and since inhibitors for collagen synthesis block the expression of osteoblast markers by these cells, it has been speculated that AsA-dependent activity is required for the synthesis of the extracellular matrix of type I collagen (Franceschi and Iyer, 1992;Franceschi et al, 1994;MacCauley et al, 1996;Takeuchi et al, 1996;Takeuchi et al, 1997;Xiao et al, 1997;Otsuka et al, 1999). However, in this study, because collagenous bone matrix had already formed before the start of experiment, and since osteoblastic cells contained abundant enlarged rER despite the absence of an osteoid layer in scorbutic rats, AsA deficiency appears to have obliterated the functions (other than collagen synthesis) of the osteoblasts.…”

“…receptor (Takeuchi et al, 1996) in osteoblastic cells. AsA is also required for osteoclast differentiation in vitro (Ragab et al, 1998;Otsuka et al, 2000).…”