Differentiation and Transforming Growth Factor-β Receptor Down-regulation by Collagen-α2β1 Integrin Interaction Is Mediated by Focal Adhesion Kinase and Its Downstream Signals in Murine Osteoblastic Cells

Abstract

Interaction of type I collagen (COL(I)) withTransient expression of a MAPK-specific phosphatase, CL100, also suppressed the elevation of ALP activity. In addition, introduction of a constitutively active MAPK kinase enhanced ALP activity in the absence of collagen production. TGF-␤ receptor down-regulation was abrogated by treatments that inactivate FAK, whereas the expression of CL100 had no effect. These results demonstrate that COL(I)-␣2␤1 integrin interaction facilitates differentiation and down-regulates TGF-␤ receptors via the activation of FAK and its diverse downstream signals. These signaling pathways may play an important role in the sequential differentiation of osteoblasts during bone formation.Cells of osteoblast lineage exert various functions depending upon their differentiation stages to maintain bone formation. Production of type I collagen (COL(I)1 ) is one of the earlier events during this process, followed by sequential expression of alkaline phosphatase (ALP) and osteocalcin. Mineralization of newly formed matrix takes place thereafter (1). To form bones with structural integrity and physical strength, it appears to be of critical importance to maintain the sequential development of these multiple osteoblastic phenotypes.Using osteoblastic cells from bone or of clonal origin, various hormones and cytokines are shown to affect the differentiation process and functional properties of these cells (1, 2). In addition to these factors, matrix COL(I) is required for the differentiation of osteoblasts (3, 4). We have demonstrated that the effect of COL(I) on the development of osteoblastic properties is mediated by the interaction of COL(I) with cell-surface ␣2␤1 integrin and that the interaction also causes down-regulation of transforming growth factor (TGF)-␤ receptors without affecting the expression of the receptor mRNA (5), suggesting the translocation of TGF-␤ receptors from cell surface to intracellular compartments. Because TGF-␤ potently stimulates matrix protein synthesis but inhibits differentiation of osteoblasts (5), the down-regulation of its receptors may serve to facilitate further maturation of osteoblasts and mineralization of newly formed matrix. However, the mechanism and the intracellular pathways that control the differentiation and TGF-␤ receptor down-regulation of osteoblastic cells by COL(I)-␣2␤1 integrin interaction remain unclear.Accumulating evidence indicates that stimulation of ␤1 integrin by matrix proteins initiates intracellular signaling pathways in many types of cells (6 -9). One of the initial events triggered by the stimulation of ␤1 integrin is the association of its cytoplasmic domain with focal adhesion kinase (FAK), a cytosolic non-receptor tyrosine kinase, followed by tyrosine phosphorylation and activation of FAK (10). FAK-deficient mice display severe defects of mesodermal development in embryogenesis (11). The phenotype of FAK-deficient mice is strikingly similar to the fibronectin-deficient phenotype (12), suggesting that FAK uniquely mediates matrix-in...

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.…”

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

“…Specifically, this interaction triggers osteoblast-specific expression of alkaline phosphatase and osteocalcin mRNAs, and ultimately, mineralization of bone tissue in a stage-specific sequence [12,13]. Matrix responsiveness during osteogenesis is caused by the aggregation of the α2β1 [9,[14][15][16] and αvβ3 [17] integrins that subsequently activate intracellular signaling cascades, resulting in the phosphorylation of the osteoblast-specific transcription factor Runx2/Cbfa-1 [15,18,19]. Understanding the molecular signaling mechanisms associated with these events and the osteogenic differentiation of hMSC as a whole, will accelerate the practical application of stem cell engineering.…”

“…How these pathways are organized in developing bone is not well known, though osteoblasts in osteoporosis or osteoarthritis patients have reduced FAK activity [24]. Inhibition of FAK by herbimycin A, cytochalasin D, or overexpression of antisense FAK mRNA prevents the expression of ERKdependent alkaline phosphatase activity in osteoblast-like cell lines, consequently preventing further osteogenic differentiation [14]. Despite these observations, the role of FAK signaling in controlling hMSC commitment to the osteogenic phenotype, rather thanmaturation of committed osteoblasts, has not been examined.…”

Filamin Associates with Smads and Regulates Transforming Growth Factor-β Signaling

“…Expression of TGF-␤ receptors is controlled by ligand-activated integrins in breast cancer (50) and osteoblastic (51,52) cells. In bleomycin-induced pulmonary fibrosis, integrin ␣ v ␤ 6 binds to latency-associated peptide and induces TGF-␤ activation to induce pulmonary inflammation (53).…”