Matrix Metalloproteinase-dependent Activation of Latent Transforming Growth Factor-β Controls the Conversion of Osteoblasts into Osteocytes by Blocking Osteoblast Apoptosis

Abstract

Upon termination of bone matrix synthesis, osteoblasts either undergo apoptosis or differentiate into osteocytes or bone lining cells. In this study, we investigated the role of matrix metalloproteinases (MMPs) and growth factors in the differentiation of osteoblasts into osteocytes and in osteoblast apoptosis. The mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts were either grown on two-dimensional (2-D) collagen-coated surfaces, where they morphologically resemble flattened, cuboidal bone lining cells, or embedded in three-dimensional (3-D) collagen gels, where they resemble dendritic osteocytes constituting a network of cells. When MC3T3-E1 osteoblasts were grown in a 3-D matrix in the presence of an MMP inhibitor (GM6001), the cell number was dose-dependently reduced by approximately 50%, whereas no effect was observed on a 2-D substratum. In contrast, the murine mature osteocyte cell line, MLO-Y4, was unaffected by GM6001 under all culture conditions. According to TUNEL assay, the osteoblast apoptosis was increased 2.5-fold by 10 M GM6001. To investigate the mechanism by which MMPs mediate the survival of osteoblasts, we examined the effect of GM6001 on MC3T3-E1 osteoblasts in the presence of extracellular matrix components and growth factors, including tenascin, fibronectin, laminin, collagenase-cleaved collagen, gelatin, parathyroid hormone, basic fibroblast growth factor, vascular epidermal growth factor, insulin-like growth factor, interleukin-1, and latent and active transforming growth factor-␤ (TGF-␤). Only active TGF-␤, but not latent TGF-␤ or other agents tested, restored cell number and apoptosis to control levels. Furthermore, we found that the membrane type MMP, MT1-MMP, which is produced by osteoblasts, could activate latent TGF-␤ and that antibodies neutralizing endogenous TGF-␤ led to a similar decrease in cell number as GM6001. Whereas inhibitors of other protease families did not induce osteoblast apoptosis, an inhibitor of the p44/42 mitogen-activated protein kinase showed the same but non-synergetic effect as GM6001. These findings suggest that MMP-activated TGF-␤ maintains osteoblast survival during trans-differentiation into osteocytes by a p44/42-dependent pathway.The skeleton is a dynamic tissue that is continuously remodeling to sustain calcium homeostasis, repair microfractures, and react to strain and stress of the skeleton. The remodeling process is a complex process and relies on the coupling between bone resorption and formation that involves osteoclasts, osteoblasts, and osteocytes.The constant regeneration of bone emphasizes the delicate balance between bone resorption and bone formation, which otherwise may lead to pathological conditions such as osteoporosis or osteopetrosis. The investigation of the cellular actions of the major players of bone remodeling may therefore contribute significantly to the discovery of new and better drugs for the treatment of osteoporosis (1).The major pharmaceutical interventions for treatment and prevention of osteop...

Basigin/CD147 Promotes Renal Fibrosis after Unilateral Ureteral Obstruction

“…Because TGF-␤ expression was upregulated after UUO to similar extents in Bsg ϩ/ϩ and Bsg Ϫ/Ϫ mice up to day 7, it is conceivable that the responsiveness to TGF-␤ is linked to the difference in renal fibrosis in Bsg ϩ/ϩ and Bsg Ϫ/Ϫ mice. In this context, it is noteworthy that both MMP-2 and MMP-9 are an activator of TGF-␤, 4,5 and that Bsg is a strong inducer of MMP family. Thus, there may be a molecular circuit between Bsg, MMPs, and TGF-␤ ( Figure 7).…”

“…3,4 Conversely, some MMPs, such as MMP-2, MMP-9, and MT1-MMP, activate latent TGF-␤. 5,6 Therefore, ECM synthesis and degradation are intricately regulated by TGF-␤, MMPs, and others. In this context, it should also be noted that hyaluronan plays a crucial role in the TGF-␤-driven differentiation of fibroblasts into myofibroblasts.…”

Matrix remodeling during endochondral ossification

“…Other studies have shown that osteoblast survival is actually regulated by MT1-MMP through activation of transforming growth factor-β (TGFβ) [52]. Interestingly, TGFβ has also been implicated in the process of differentiation of hypertrophic chondrocyte through the upregulation of parathyroid-hormone-related protein (PTHrP) [53].…”

“…Later in development and subsequent to the delay in secondary-site ossification, proliferation of chondrocytes in the growth plate at the primary site is impaired. The progressive osteopenia observed in these mice results from a decrease in bone deposition, as well as an increase in bone resorption, with a high number of osteoclasts at the chondro-osseous junction.Other studies have shown that osteoblast survival is actually regulated by MT1-MMP through activation of transforming growth factor-β (TGFβ) [52]. Interestingly, TGFβ has also been implicated in the process of differentiation of hypertrophic chondrocyte through the upregulation of parathyroid-hormone-related protein (PTHrP) [53].…”

Apoptosis of human endothelial cells is accompanied by proteolytic processing of latent TGF-β binding proteins and activation of TGF-β

“…10,15 The activation of TGF-b by the matricellular protein trombospondin-1, 16 as well as by the integrin a v b 6 17,18 seem to proceed via similar mechanisms. In addition, a number of proteases including plasmin, 19,20 matrix metalloproteinases MMP-3, -9, and MT1-MMP, 21,22 as well as exocytosed cathepsin B 23 have been identified as activators of latent TGF-b, at least in vitro.…”