Cytoskeletal reorganisation, 1α,25-dihydroxy vitamin D3 and human MG63 osteoblast maturation

Abstract

Bone tissue is especially receptive to physical stimulation and agents with the capacity to mimic the signalling incurred via mechanical loading on osteoblasts may find an application in a bone regenerative setting. Recently this laboratory revealed that the major serum lipid, lysophosphatidic acid (LPA), cooperated with 125-dihydroxy vitamin D3 (D3) in stimulating human osteoblast maturation. Actin stress fiber accrual in LPA treated osteoblasts would have generated peripheral tension which in turn may have heightened the maturation response of these cells to D3. To test this hypothesis we examined if other agents known to trigger stress fiber accumulation cooperated with D3 in stimulating human osteoblast maturation.Colchicine, nocodazole and LPA all co-operated with D3 to promote MG63 maturation in a MEK dependent manner. In contrast, calpeptin, a direct activator of Rho kinase and stress fiber accumulation did not act with D3 to secure MG63 differentiation. Herein we describe how the signalling elicited via microtubule disruption cooperates with D3 in the development of mature osteoblasts.

24,25-Dihydroxyvitamin D3 cooperates with a stable, fluoromethylene LPA receptor agonist to secure human (MG63) osteoblast maturation

“…LPA has been found to stimulate expression of Fra-2, albeit for rodent fibroblasts, consequent to MEK activation [38]. In our hands we consistently find that the synergistic increase in ALP following co-stimulation with VDR agonists and other factors is always MEK dependent [14,15,18].…”

“…What remains to be determined is whether 24R,25D can promote hOB maturation when co-administered with agents known to synergistically co-operate with 1,25D; it is becoming clear that 1,25D often needs to interact with other factors to prosecute the desired response in target cells [13]. In our hands we consistently find that hOBs do not mobilise alkaline phosphatase (ALP) when treated with 1,25D in a serum-free in vitro setting and will only do so when the cells are in receipt of both 1,25D and certain growth factors such as epidermal growth factor [14], lysophosphatidic acid (LPA) or certain LPA receptor selective agonists [15][16][17][18]. Whilst a significant body of work is emerging on the role of LPA in osteoblast, and indeed skeletal biology in general, we will not expand on those areas here.…”

Chitinase 3-like 1 expression by human (MG63) osteoblasts in response to lysophosphatidic acid and 1,25-dihydroxyvitamin D3

“…Similar results were obtained for cells cotreated with 1,25D and EGF [34] and 1,25D and colchicine [41]. Herein we now find that During the late stages of human macrophage differentiation, Rehli and colleagues [44] found YKL-40 to be markedly enhanced.…”

Cell cytoskeletal changes effected by static compressive stress lead to changes in the contractile properties of tissue regenerative collagen membranes

“…Therefore, any stiffening of the external matrix is being reflected in real-time stiffening of the internal cytoskeleton. Both cell survival and osteogenic maturation have been linked to cytoskeletal pre-stress and the rigidity of the substrate, which are both properties modulated by the dense collagen environment (Mansell et al, 2009;Nieponice et al, 2007). Conventionally, one would consider that dense collagen acts to enhance RhoA through flattening of the cell and the provision of a stiff environment, which in turn promotes osteogenic differentiation, as has been demonstrated by experiments on human MSCs (Chen et al, 2011;Hamamura et al, 2012).…”