gms | German Medical Science

Introduction:

Preclinical studies on the application of BMP2 on bone healing were very promising. Although, the clinical data fell below expectations and unwanted side effects were reported. For these reasons, it is desirable to improve future clinical usage through a reduction of BMP2 concentrations yet maintaining positive function. Nitric oxide (NO) has been a candidate in several investigations for the treatment of bone malignancies [1, 2]; furthermore, there has been some connections between the BMP2 and NO pathway suggested [3]. The aim of the present study is to determine if the NO pathway is able to mediate BMP2 activity.

Methods: BMP2 activity is quantified by use of a stable luciferase reporter cell line system C2C12BreLuc; composed of an ID1-promoter (SMAD binding) fused to luciferase. Following starvation, cells were stimulated for 6 or 24 hours in different combinations of: rhBMP2 1nM (Osteogenetics - E-coli); DetaNONOate (NO Donor) 1 - 500µM; L-Arginine (NO Substrate) 0.1 - 2.5mM; LNAME (NO Synthase (NOS) inhibitor) 1 - 1000µM. ID1 reporter activity was measured by luciferase assay and normalized to total protein content. Statistics: Kruskal-Wallis tests, Mann-Whitney-U Test, p < 0.05

Results: The NO substrate L-Arginine alone had no effect on ID1 reporter activity at 6 and 24 hours, whereas BMP2 increased significantly ID1 activity. The combination of L-Arginine and BMP2 did not enhance reporter activity at 6 hours. At 24 hours, however, ID activity was significantly higher after L-Arginine and BMP2 application compared to BMP alone. L-Arginine's supplementary effect on 1nM BMP2 signalling was similar to a concentration of 5nM BMP2 without L-Arginine. The application of LNAME (inhibitor of NO Synthase) was able to significantly reduce ID1 reporter activity induced by L-Arginine combined with BMP2. The addition of the NO donor DetaNONOate in combination with BMP2 for 24 hours significantly improved reporter activity compared against BMP2 alone.

Conclusion: The study has shown that the NO pathway is able to modulate BMP2 signalling. L-Arginine's supplementary effects of BMP2 activity occur in a time dependent manner. We confirmed L-Arginine's additive effects on BMP2 activity to be a result of NO production through the addition of the NOS inhibitor, LNAME, which led to loss of L-Arginine's supplementary effects. The NO pathway modulation of BMP2 activity is further underlined by direct NO addition leading to an increase of BMP2 activity. The study clearly demonstrated the ability for NO to improve the activity of BMP2, which might be a future option to treat patients with reduced concentrations of BMP2 to prevent side effects.