Monday, September 26, 2005
9360

PSEF 2005 Scientific Essay Contest D. Ralph Millard, MD Investigator Award, First Place: siRNA Mediated Noggin Suppression Enhances Osteoblast Differentiation and Mineral Deposition

Derrick Wan, MD, Jason H. Pomerantz, Randall P. Nacamuli, and Michael T. Longaker, MD.

The BMP antagonist Noggin is differentially expressed in the developing murine cranial vault, with elevated expression found in the mesenchyme and dura mater of sutures destined to remain patent. While it is presumed that decreased Noggin leads to an increase in osteogenesis – and eventual fusion of the posterior-frontal suture – the effect of altered Noggin expression on osteoblast development and subsequent suture fate remains unknown. We show that Noggin may play a crucial role in the regulation of differentiation and thus potentially dictate the rate of osteogenesis. A restriction enzyme-generated system was employed to create 5 individual noggin directed siRNA's, 2 of which possessed sufficient capacity to abrogate the detection of protein. Suppression of noggin resulted in enhanced mineralization and bone nodule deposition as assessed by Alizarin red and von Kossa staining following 21 days of differentiation. Quantitative Real-Time RT-PCR analysis of gene expression at this same time point revealed elevated transcript levels for both runx2/cbfal and osteopontin when compared to undifferentiated cells. This upregulation was noted to be smaller, however, relative to normal differentiated controls suggesting a more terminally differentiated osteoblast phenotype in the absence of noggin. In accordance with this, expression of a late marker for osteoblast differentiation, osteocalcin, demonstrated the greatest rise when noggin protein was abrogate. Our results imply that Noggin may serve to naturally regulate osteoblast differentiation, with elimination of this protein leaving BMP activity unopposed to drive osteogenesis. Such an absence may presumable contribute to pathologic suture fusion and suggest a future avenue for therapy to potentially modify postnatal skeletal development.