gms | German Medical Science

Objectives: Smoking has negative effects on bone integrity, and is associated with increased incidence of fractures. Following orthopedic surgeries smokers show delayed fracture healing and increased frequency of complications, resulting in prolonged hospital stays. One crucial factor responsible for fracture repair is the recruitment and differentiation of mesenchymal stem cells (MSCs). Bone morphogenetic proteins (BMPs) are one of the most compelling cytokines in bone for their major role in the osteogenic differentiation of MSCs. Our aim was to investigate the effect of cigarette smoke on BMP signaling in MSCs and to evaluate at which step the pathway is affected.

Methods: Human immortalized MSCs (SCP-1 cells) were treated with cigarette smoke extract (CSE) concentrations associated with smoking up to 20 cigarettes/day. BMP signaling was analyzed using an adenovirus-based reporter assay system expressing luciferase under the control of the Smad1/5/4 responsive element. Primary cilia and BMP signaling were analyzed by Western blot and immunofluorescence staining. BMP-9 levels were quantified by ELISA. Human samples were obtained in accordance with the ethical vote of the University Hospital Tübingen and with patients' written consent. Data were analyzed using Kruskal-Wallis H-test

Results and conclusion: BMP-2 and BMP-9 significantly induce BMP signaling in SCP-1 cells. However, induction through Activin receptor like kinase (Alk) 1 (BMP-9 receptor) is significantly stronger than through Alk6 (BMP-2 receptor). CSE exposure caused a significant reduction in the BMP signaling (p<0.001). Interestingly, the pathway thought BMP-9 receptor was less affected. CSE exposure disrupts structure of primary cilia, sensory organelles proposed to be involved in BMP signaling. In line, disruption of primary cilia by chloral hydrate significantly reduced BMP signaling (p<0.001). Despite inhibition of BMP signaling, protein levels of phospho-Smad1/5 (active form) did not change after CSE exposure. However, incubation with CSE significantly reduced translocation of phospho-Smad1/5/4 complex into the nucleus, affecting expression of BMP target genes involved in osteogenesis (Fig). BMP-9 serum levels are significantly lower in smokers than non-smokers (p<0.05) comparable to patients with delayed fracture healing.

In summary, inhibition of BMP signaling by CSE was closely associated with impaired nuclear translocation of phospho-Smad1/5/4 complex and impaired osteogenic differentiation. BMP-9 was less affected than BMP-2. Thus, low BMP-9 levels found in blood from smokers could be responsible for delayed fracture healing.