Abstract
BackgroundBone morphogenic proteins (BMPs) play a key role in bone formation. Consequently, it was expected that topical application of recombinant human (rh)BMP-2 and rhBMP-7 would improve the healing of complex fractures. However, up to 36% of fracture patients do not respond to this therapy. There are hints that a systemic increase in transforming growth factor β1 (TGFβ1) interferes with beneficial BMP effects. Therefore, in the present work we investigated the influence of rhTGFβ1 on rhBMP signaling in primary human osteoblasts, with the aim of more specifically delineating the underlying regulatory mechanisms.MethodsBMP signaling was detected by adenoviral Smad-binding-element-reporter assays. Gene expression was determined by reverse transcription polymerase chain reaction (RT-PCR) and confirmed at the protein level by western blot. Histone deacetylase (HDAC) activity was determined using a test kit. Data sets were compared by one-way analysis of variance.ResultsOur findings showed that Smad1/5/8-mediated rhBMP-2 and rhBMP-7 signaling is completely blocked by rhTGFβ1. We then investigated expression levels of genes involved in BMP signaling and regulation (for example, Smad1/5/8, TGFβ receptors type I and II, noggin, sclerostin, BMP and activin receptor membrane bound inhibitor (BAMBI), v-ski sarcoma viral oncogene homolog (Ski), Ski-related novel protein N (SnoN) and Smad ubiquitination regulatory factors (Smurfs)) and confirmed the expression of regulated genes at the protein level. Smad7 and SnoN were significantly induced by rhTGFβ1 treatment while expression of Smad1, Smad6, TGFβRII and activin receptor-like kinase 1 (Alk1) was reduced. Elevated SnoN expression was accompanied by increased HDAC activity. Addition of an HDAC inhibitor, namely valproic acid, fully abolished the inhibitory effect of rhTGFβ1 on rhBMP-2 and rhBMP-7 signaling.ConclusionsrhTGFβ1 effectively blocks rhBMP signaling in osteoblasts. As possible mechanism, we postulate an induction of SnoN that increases HDAC activity and thereby reduces the expression of factors required for efficient BMP signaling. Thus, inhibition of HDAC activity may support bone healing during rhBMP therapy in patients with elevated TGFβ serum levels.
Highlights
Bone morphogenic proteins (BMPs) play a key role in bone formation
We demonstrated that transforming growth factor b1 (TGFb1) inhibits rhBMP-2 and rhBMP-7 signaling in primary human osteoblasts [14]
Human recombinantTGFb1, rhBMP-2 and rhBMP-7 were from Peprotech (London, UK); cell culture medium and supplements were from PAA (Cölbe, Germany); primary and secondary antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany) and Cell Signaling (Frankfurt am Main, Germany); chemicals were from Sigma (Munich, Germany)
Summary
Bone morphogenic proteins (BMPs) play a key role in bone formation. it was expected that topical application of recombinant human (rh)BMP-2 and rhBMP-7 would improve the healing of complex fractures. While bone is formed by osteoblasts, which are of mesenchymal origin, it is resorbed by osteoclasts that are derived from the hematopoietic system An imbalance in this process may cause pathological loss of bone mass as seen with delayed fracture healing, osteoporosis and other metabolic bone diseases. Clinical trials using rhBMPs were successful in the treatment of open tibial fractures, distal tibial fractures, tibial non-unions, scaphoid non-unions and atrophic long bone non-unions [2,3,4,5,6,7] Despite these proven positive effects of BMPs on bone healing, the universal use of rhBMPs is tempered by high costs, lingering safety concerns (for example, vertebral osteolysis, ectopic bone formation, radiculitis or cervical soft tissue swelling), and a relatively high failure rate with up to 36% of patients not responding to this therapy [8,9]. In order to design an improved second-generation therapy, it is necessary to fully understand the molecular mechanisms of the activity of rhBMPs in the setting of bone defect therapy
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