Cigarette Smoke Induces the Risk of Metabolic Bone Diseases: Transforming Growth Factor Beta Signaling Impairment via Dysfunctional Primary Cilia Affects Migration, Proliferation, and Differentiation of Human Mesenchymal Stem Cells

Abstract

It is well established that smoking has detrimental effects on bone integrity and is a preventable risk factor for metabolic bone disorders. Following orthopedic surgeries, smokers frequently show delayed fracture healing associated with many complications, which results in prolonged hospital stays. One crucial factor responsible for fracture repair is the recruitment and differentiation of mesenchymal stem cells (MSCs) at early stages, a mechanism mediated by transforming growth factor β (TGF-β). Although it is known that smokers frequently have decreased TGF-β levels, little is known about the actual signaling occurring in these patients. We investigated the effect of cigarette smoke on TGF-β signaling in MSCs to evaluate which step in the pathway is affected by cigarette smoke extract (CSE). Single-cell-derived human mesenchymal stem cell line (SCP-1 cells) were treated with CSE concentrations associated with smoking up to 20 cigarettes a day. TGF-β signaling was analyzed using an adenovirus-based reporter assay system. Primary cilia structure and downstream TGF-β signaling modulators (Smad2, Smad3, and Smad4) were analyzed by Western blot and immunofluorescence staining. CSE exposure significantly reduced TGF-β signaling. Intriguingly, we observed that protein levels of phospho-Smad2/3 (active forms) as well as nuclear translocation of the phospho-Smad3/4 complex decreased after CSE exposure, phenomena that affected signal propagation. CSE exposure reduced the activation of TGF-β modulators under constitutive activation of TGF-β receptor type I (ALK5), evidencing that CSE affects signaling downstream of the ALK5 receptor but not the binding of the cytokine to the receptor itself. CSE-mediated TGF-β signaling impaired MSC migration, proliferation, and differentiation and ultimately affected endochondral ossification. Thus, we conclude that CSE-mediated disruption of TGF-β signaling in MSCs is partially responsible for delayed fracture healing in smokers.

Primary Human Chondrocytes Affected by Cigarette Smoke—Therapeutic Challenges

“…Aspera-Werz et al reported that although nicotine and cotinine did not directly induce ROS, they impaired the anti-oxidative defense mechanisms within mesenchymal stem cells by inhibiting anti-oxidative enzyme activity [44]. In our study, ROS production was generated significantly when chondrocytes were exposed to 10% CSE, which is in line with smoking approximately 20 cigarettes (1 pack) a day [40]. As chondrocytes are normally in a quiescent condition and only proliferate once activated, the survival and death of chondrocytes are vital for the preservation of articular cartilage [14].…”

“…Toxins contained in cigarettes have been shown to induce oxidative stress [2], inflammatory responses [38], or hypoxia [39], which all can damage cartilage. Recently, we reported that CSE negatively affects the migration, proliferation, and chondrogenic differentiation of mesenchymal stem cells through impairing the transforming growth factor-β (TGF-β) signaling [40]. However, the effects of cigarette smoke on primary human chondrocytes remain unclear.…”

Assessment of tobacco heating system 2.4 on osteogenic differentiation of mesenchymal stem cells and primary human osteoblasts compared to conventional cigarettes

“…Nuclei were stained with Hoechst 33342 (1:1000). Images were taken with an epifluorescence microscope (EVOS FL, life technologies, Darmstadt, Germany), and primary cilia length was analyzed using the ImageJ software (Version 1.5, NIH, Bethesda, MD, United States) (line tool) based on the maximum intensity projection method[ 39 , 40 ].…”

Feasibility of Cell Lines for In Vitro Co-Cultures Models for Bone Metabolism

“…Alkaline phosphatase (ALP) activity was significantly higher in co-cultures with SaOS-2 cells, which have a very high basal ALP activity [205], than in co-cultures with SCP-1 cells, which normally show an increase in ALP activity with differentiation [206] (Figure 5C). Consequently, after 14 days of culture formation of mineralized matrix was also higher in co-cultures with SaOS-2 cells than in co-cultures with SCP-1 cells ( Figure 5D).…”