Enhancement of bone morphogenetic protein-2 expression and bone formation by coumarin derivatives via p38 and ERK-dependent pathway in osteoblasts.

Osteoporosis is a reduction in skeletal mass due to an imbalance between bone resorption and bone formation. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. Therefore, components involved in BMP activation are good targets for the development of anti-osteoporosis drugs. In this study, imperatorin and bergapten, two coumarin derivatives, were shown to enhance alkaline phosphatase (ALP) activity, type I collagen synthesis and bone nodule formation in primary cultured osteoblasts. Imperatorin and bergapten increased mRNA levels of BMP-2 using quantitative RT-PCR, whereas the BMP-2 antagonist noggin attenuated the increase of ALP activity induced by imperatorin and bergapten, indicating that BMP-2 expression is required for the action of imperatorin and bergapten in osteoblastic maturation. Both imperatorin and bergapten enhanced the phosphorylation of SMAD (transcription factors activated by TGF-beta) 1/5/8, p38 and extracellular signal-regulated protein (ERK). Pretreatment of osteoblasts with p38 inhibitor (SB203580) or mitogen-activated protein kinase inhibitor (PD98059) or transfected with dominant negative mutant of p38 or ERK antagonized the elevation of BMP-2 expression and ALP activity induced by imperatorin and bergapten. Local administration of imperatorin or bergapten into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the BMP-2 immunostaining and bone volume of secondary spongiosa in tibia. Taken together, our results provide evidence that coumarin derivatives increase BMP-2 expression and enhance bone formation in rat via the p38 and ERK-dependent signaling pathway.

Inhibitory effect of the plant-extract osthole on L-type calcium current in NG108-15 neuronal cells.

The effects of osthole, a coumarin isolated from Cnidium monnieri (L.) Cusson, on ionic currents in a mouse neuroblastoma and rat glioma hybrid cell line, NG105-18, were investigated with the aid of the whole-cell voltage-clamp technique. Osthole (0.3-100 microM) caused an inhibition of voltage-dependent L-type Ca(2+) current (I(Ca,L)) in a concentration-dependent manner. Osthole produced no change in the overall shape of the current-voltage relationship of I(Ca,L). The IC(50) value of the osthole-induced inhibition of I(Ca,L) was 4 microM. The presence of osthole (3 microM) shifted the steady state inactivation curve of I(Ca,L) to a more negative potential by approximately -15mV. Osthole (3 microM) also produced a prolongation in the recovery of I(Ca,L) inactivation. Although osthole might suppress phosophodiesterases to increase intracellular adenosine-3',5'-cyclic monophosphate (cyclic AMP) or guanosine-3',5'-cyclic monophosphate (cyclic GMP), sp-cAMPS did not affect I(Ca,L) and 8-bromo-cyclic GMP slightly suppressed it. Thus, osthole-mediated inhibition of I(Ca,L) was not associated with intracellular cyclic AMP or GMP. However, no effect of osthole on voltage-dependent K(+) outward current was observed. Under a current-clamp mode, osthole could decrease the firing frequency of action potentials. Therefore, the channel-blocking properties of osthole may, at least in part, contribute to the underlying mechanisms by which it affects neuronal or neuroendocrine function.