Up-regulation of alkaline phosphatase expression in human primary osteoblasts by cocultivation with primary endothelial cells is mediated by p38 mitogen-activated protein kinase-dependent mRNA stabilization.

For the regeneration of bone in tissue engineering applications, it is essential to provide cues that support neovascularization. This can be achieved by cell-based therapies using mature endothelial cells (ECs) or endothelial progenitor cells. In this context, ECs were used in various in vivo studies in combination with primary osteoblasts to enhance neovascularization of bone grafts. In a previous study, we have shown that cocultivation of human primary ECs and human primary osteoblasts (hOBs) leads to a cell contact-dependent up-regulation of alkaline phosphatase (ALP) expression in osteoblasts, indicating that cocultivated ECs may support osteogenic differentiation and osteoblastic cell functions. In the present study, we investigated this effect in more detail, revealing a time and cell number dependency of EC-mediated up-regulation of the early osteoblastic marker ALP, whereas osteocalcin, a late marker of osteogenesis, was down-regulated. The effect on ALP expression was bidirectional specific for both cell types. Functional inhibition of gap junctional communication between ECs and hOBs by 18alpha-glycyrrhetinic acid had only a weak suppressive effect on EC-mediated ALP up-regulation. In contrast, inhibition of p38 mitogen-activated protein kinase nearly completely prevented the EC-mediated stimulation of osteoblastic ALP expression. To investigate the molecular mechanism underlying the ALP up-regulation, we examined the effect of EC cocultivation on osteoblastic ALP promoter activity as well as mRNA stability. Cocultivation of ECs with hOBs significantly elevated the half-life of osteoblastic ALP mRNA without affecting its promoter activity. In summary, our data show that EC-mediated up-regulation of osteoblastic ALP expression is cell-type specific and is posttranscriptionally regulated via p38 mitogen-activated protein kinase-dependent mRNA turn-over.