Thrombospondin co-localises with TGFβ and IGF-I in the extracellular matrix of human osteoblast-like cells and is modulated by 17β estradiol

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Abstract
Thrombospondin (TSP) is a multifunctional glycoprotein which is synthesised by several cell types including osteoblasts, and incorporated into the extracellular matrix (ECM) of these cells. The function and regulation of TSP in bone is not clear. In this study, using a long term culture model of human osteoblast-like cells, we examined the distribution of TSP in the ECM and its modulation by added estradiol. In this model the osteoblast-like cells form a regular multilayer which continues to increase in depth up to 50 days post confluence. In the ECM of these cultures and in 19-week fetal bone, the bone markers osteocalcin and alkaline phosphatase were diffusely distributed in the matrix. In contrast, labelling for TSP was concentrated, confined to the banded collagen and its immediately adjacent ECM. This pattern of labelling resembled that of the growth factors transforming growth factorβ-I (TGFβ), and insulin-like growth factor-I (IGF-I), with which TSP label co-localised. Labelling intensities were comparable between fetal bone and the in vitro material for TSP, TGFβ and IGF-I. TSP label was present by 10 days post confluence, reached a maximum by 20 days, and declined slowly thereafter, a time course which was similar to that of IGF-I. Incubation of osteoblast-like cell cultures with 17β estradiol resulted in an increase in multilayer depth and a maximal 3-fold increase in TSP labeling at 30 days as well as approximately 2-fold increases for TGFβ and IGF-I. The dose-response relationship for these responses to estradiol treatment was biphasic with maximal increases at 10−10 M–10−11 M of added estradiol. Treatment with 17α estradiol produced labelling intensities that were not significantly different from controls. Studies with other cell types have suggested that TSP may be involved in modulation of growth factor activity. The similarities between TSP, TGFβ and IGF-I, in terms of their distribution and regulation by 17β estradiol treatment, may indicate a role for TSP in modulating bone cell proliferation and function through interaction with local growth factors.