We have found that heparanase (HPSE) stimulates human osteoblast cell growth. This study explored the mechanism of HPSE in the stimulation of osteoblasts from osteoporotic and healthy subjects. We further hypothesized that the structure of chromatin is modified by HPSE via activating phosphorylation of histone H3.

Osteoblast primary cell cultures originating from osteoporotic and healthy human subjects (n=8/group) were developed and exposed to exogenous HPSE at a series of concentrations. The mineralized nodules were stained using Alizarin red-S, a dye-binding ~2 mole of Ca2+/mol in solution, and then calcium mineral content was quantified by measuring the amount of AR-S bound to mineralized nodules in the cultures. A RT-qPCR array assay was used to detect osteogenic gene expression on osteoblasts after treatment with HPSE. A flow cytometry approach was used for the detection of histone H3 modification.

Heparanase significantly increased the calcium content of osteoblasts cultures from healthy and osteoporotic subjects at day 14, but the effect on osteoporotic cells was much greater. Osteoblasts exposed to heparanase at concentrations of 6, 3, 1, and 0.1μg/mL yielded 257, 222, 145, and 134% increase in the calcium content in osteoporotic subjects; and 160, 152, 121 and 106% increase in healthy subjects. A number of osteogenic genes were dramatically up-regulated by the exogenous HPSE. In the osteoporotic subjects, BMPs, COLs, GDF10, COMP (skeletal development and bone mineral metabolism), ENAM (bone mineralization) and ALPL (ossification) were significantly up-regulated. Genes involved in cell growth, IGF1, IGF2, VEGF, CSF2, CSF3 and growth factors’ receptors such as FGFR1, FGFR2, EGFR, TGFBR1, and cell adhesion molecules, CTSK, VCAM, BGN and CD36 were also up-regulated. In the healthy subjects, FGF, CSF2, BMP2, ALPL, and ECM protease (MMP9) were up-regulated, while ENAM and FGFR2 were down-regulated. In addition, HPSE modified the structure of chromatin by increasing positive events of histone H3 phosphorylation (91%) compared to control cultures (2.4%) from osteoporotic subjects, and 39% vs. 23% in healthy subjects. HPSE markedly promotes mineralization of osteoblasts in osteoporotic and healthy subjects.

HPSE up-regulates osteogenic gene expression, correlates with markers of bone formation, activates histone H3 phosphorylation, and seems to be playing a role in bone remodeling at steps preceding BMP, VEGF and ALPL’s during osteoblast cell growth.