Synthesis of collagenase and collagenase inhibitors by osteoblast-like cells in culture

Abstract

A rat osteosarcoma cell clone (ROS 17/2), and osteoblast-enriched populations from rat calvaria cultured in the presence of concanavalin A, have been shown to produce latent collagenase and collagenase inhibitors. The enzymes and inhibitor activities from the ROS 17/2 cells were concentrated by ammonium sulphate precipitation and separated by gel filtration on AcA 54 resin. The size of the latent collagenase (Mr z 58000) was reduced on conversion to active enzyme ( M , ~448000) by p-aminophenylmercuric acetate. Latent and active forms of gelatinase activity, similar in size to the corresponding forms of collagenase, were also resolved. The collagenase inhibitor activity, which was sensitive to organomercurials, was recovered in two peaks ( M , ~668000 and 30000). The active collagenase cleaved interstitial collagens (type I = I11 > 11) producing typical 3/4 and 1/4 fragments. This activity was inhibited by the metal ion chelators ethylenediaminetetraacetic acid and o-phenanthroline. Additional specific cleavages of native collagen were also observed which, from the susceptibility of this activity to phenylmethylsulphonyl fluoride, leupeptin and antipain, suggested the presence of a second collagenolytic enzyme. This synthesis of collagenolytic enzymes by these osteoblast-like cells suggests that individual osteoblasts, like fibroblasts, are capable of both synthesizing and degrading their respective organic matrices in vivo.Collagenases that characteristically cleave native interstitial collagens at neutral pH are produced by a variety of connective tissues and have been implicated in the physiological and pathological breakdown of collagen [I -31. However, whereas fibroblasts have been shown to produce collagenase in soft connective tissues, .the cellular origin of the collagenase produced by bone tissues [4-61 is equivocal. Earlier studies on bone cells derived from foetal rat calvariae have failed to demonstrate collagenase synthesis by any of several distinct populations of cells [7], which are believed to contain osteoblast-like and osteoclast-like cells [S]. However, the level of collagenase required to effect physiological remodelling of collagen could conceivably be below the detectable range of the most sensitive assays. A number of methods have been used to stimulate collagenase production by connective tissue cells to detectable levels. These include the addition of phorbol myristic acetate, cytochalasin B, bacterial endotoxins, soluble factors produced by monocytes, and collagen (reviewed by Biswas and Dayer [9]). In recent work we have shown that lectins will also stimulate collagenase production by gingival fibroblasts [lo, 111.Clonal populations of osteoblast-like cells have been isolated from foetal rat calvariae [12] and from a rat osteosarcoma [I 31. One of the osteosarcoma clones (ROS 17/2) has a stable osteoblast phenotype and has been shown to produce bone when transplanted into rats in vivo [13]. We have found that both this cell line and concanavalin-A-stimulated osteoblast-like population...

MMP-1 and TIMP-1 production in MG-63 cells stimulated withPrevotella nigrescenslipopolysaccharide

“…This constitutive MMP-1 seems to serve as a remodeling element of connective tissue under physiological conditions 27) . Otsuka et al also demonstrated that rat osteosarcoma cell line produced collagenolytic enzymes 23) . MMP-1 mRNA was highly expressed in this study after 48 h stimulated with Prevotella nigrescens LPS, and the increase was dosedependent.…”

“…In this study, TIMP-1 was expressed by MG-63 osteoblastic cells. Otsuka 23) , Johansen 32) , Uchida 33) . also reported that osteoblastic cells synthesized TIMP-1.…”

“…Numerous studies have examined MMPs secreted from periodontal ligament fibroblast [20][21][22] and correlated the level of expression with the invasive ability of the disease 4,6) . Individual osteoblasts, like fibroblasts, are capable of both synthesizing and degrading their respective organic matrices in vivo 23) . MMPs and TIMPs are known to be important in homeostatic matrix remodeling by osteoblasts.…”

Matrix metalloproteinases MMP‐2, ‐9 and tissue inhibitors TIMP‐1, ‐2 expression and secretion by primary human osteoblast cells in response to titanium, zirconia, and alumina ceramics

“…25,26 This protease is also secreted by osteoclasts in sub-osteoclastic zones at sites of bone resorption. 18,29,30 Despite the numerous studies undertaken on osteoblast/biomaterial interactions, the regulation of their proteolytic activity and ECM remodeling remains to be understood. Furthermore, little is known on the influence of biomaterials such as titanium or ceramics on the expression and synthesis of MMPs and TIMPs by human osteoblastic cells.…”

“…27,28 The production of MMPs by bone cells in in vivo and in vitro investigations suggests their importance in bone remodeling. 18,29 In this process, collagenases producing osteoblasts have a major role by degrading the osteoid layer, exposing the underlying mineralized matrix to osteoclastic activity. 12,30 Osteoblasts produce collagenase in response to 1,25(OH) 2 vitamin D 3 or interleukin-1 stimulation.…”