Abstract
The human cystein-rich protein 61 (hCYR61) belongs to an emerging family of genes which modulate growth and differentiation. Previously, hCYR61 was identified by us as a fast and transiently 1,25(OH)2-vitamin D3 responsive gene product in human osteoblasts by differential display PCR. Here, we further studied the role of the protein in human osteoblasts.
Using the human hFOB cell line hCYR61 mRNA was analysed by northern hybridisation. Protein levels were detected using western blotting. Intracellular localisation of the hCYR61 protein was determined using the expression as a fusion protein with green fluorescent protein. Immunohistology was performed in hFOB cells as well as primary human osteoblasts and human bone samples.
From northern analyses the hCYR61 mRNA was regulated by 1,25(OH)2-vitamin D3 as well as the growth factors TNFa, EGF, bFGF and IL1b 5-10-fold within 1 hour in the hFOB cell line. Here we show that the same factors markedly upregulated the hCYR61 protein within 24 to 48 hours in hFOB cells as has been analysed by western blotting. From cellular supernatants a highly upregulation of the hCYR61 protein by the growth factors was observed. A full length hCYR61 protein fused to the green fluorescent protein localised to the Golgi-apparatus. From immunohistology proliferating hFOB cells and primary osteoblasts express significant hCYR61 protein, whereas differentiated osteoblasts display a marked downregulation of hCYR61. In human bone high levels of hCYR61 were observed at the human growth plate as well as on surfaces of mineralised structures.
In summary, hCYR61 in human bone represents an immediate early regulated gene. The secreted protein plays a role as an extracellular matrix signaling protein which could play an important role in cell-cell communication within the bone microenvironment. The high expression level and regulation patterns observed in our studies suggest an important role in situations of bone repair and remodeling.
