Alcohol exposure decreases osteopontin expression during fracture healing and osteopontin-mediated mesenchymal stem cell migration in vitro (original) (raw)
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2012
Background: Alcohol abuse is a risk factor for bone damage and fracture-related complications. Through precise b-catenin signaling, canonical Wnt signaling plays a key role in fracture repair by promoting the differentiation of new bone and cartilage cells. In this study, we examined the effects of alcohol on the Wnt pathway in injured bone using a murine model of alcohol-induced impaired fracture healing. Methods: Male C57Bl/6 or T cell factor (TCF)-transgenic mice were administered 3 daily intraperitoneal doses of alcohol or saline. One hour following the final injection, mice were subjected to a stabilized, mid-shaft tibial fracture. Injured and contralateral tibias were harvested at 6, 9, or 14 days post-fracture for the analysis of biomechanical strength, callus tissue composition, and Wnt/b-catenin signaling. Results: Acute alcohol treatment was associated with a significant decrease in fracture callus volume, diameter, and biomechanical strength at day 14 post-fracture. Histology revealed an alcoholrelated reduction in cartilage and bone formation at the fracture site, and that alcohol inhibited normal cartilage maturation. Acute alcohol exposure caused a significant 2.3-fold increase in total b-catenin protein at day 6 and a significant decrease of 53 and 56% at days 9 and 14, respectively. lacZ staining in b-galactosidase-expressing TCF-transgenic mice revealed spatial and quantitative differences in Wntspecific transcriptional activation at day 6 in the alcohol group. Days 9 and 14 post-fracture showed that acute alcohol exposure decreased Wnt transcriptional activation, which correlates with the modulation of total b-catenin protein levels observed at these time points. Conclusions: Acute alcohol exposure resulted in significant impairment of fracture callus tissue formation, perturbation of the key Wnt pathway protein b-catenin, and disruption of normal Wntmediated transcription. These data suggest that the canonical Wnt pathway is a target for alcohol in bone and may partially explain why impaired fracture healing is observed in alcohol-abusing individuals.
Journal of Biological Chemistry, 2015
Background: Alcohol (EtOH) exposure has detrimental effects on fracture healing. Results: EtOH inhibits TGF-1 protein expression via interference with the transcription factor myeloid zinc finger 1. Conclusion: EtOH-induced deficient fracture healing may be related to reduced TGF-1. Significance: Further understanding of the mechanisms responsible for the effects of EtOH are crucial for the development of interventions aimed at averting morbidities related to EtOH consumption. Alcohol (EtOH) intoxication is a risk factor for increased morbidity and mortality with traumatic injuries, in part through inhibition of bone fracture healing. Animal models have shown that EtOH decreases fracture callus volume, diameter, and biomechanical strength. Transforming growth factor 1 (TGF-1) and osteopontin (OPN) play important roles in bone remodeling and fracture healing. Mesenchymal stem cells (MSC) reside in bone and are recruited to fracture sites for the healing process. Resident MSC are critical for fracture healing and function as a source of TGF-1 induced by local OPN, which acts through the transcription factor myeloid zinc finger 1 (MZF1). The molecular mechanisms responsible for the effect of EtOH on fracture healing are still incompletely understood, and this study investigated the role of EtOH in affecting OPN-dependent TGF-1 expression in MSC. We have demonstrated that EtOH inhibits OPN-induced TGF-1 protein expression, decreases MZF1-dependent TGF-1 transcription and MZF1 transcription, and blocks OPN-induced MZF1 phosphorylation. We also found that PKA signaling enhances OPN-induced TGF-1 expression. Last, we showed that EtOH exposure reduces the TGF-1 protein levels in mouse fracture callus. We conclude that EtOH acts in a novel mechanism by interfering directly with the OPN-MZF1-TGF-1 signaling pathway in MSC. Alcohol intoxication is a significant risk factor associated with traumatic injury. Nearly half of patients with traumatic injuries who present to emergency departments test positive for blood alcohol content (1), and intoxication is associated with an increased risk of mortality among trauma patients (2). Among the numerous physiological effects of alcohol exposure, it has been widely shown that alcohol interferes with bone formation and fracture healing. Patients who abuse alcohol have prolonged healing time following transverse tibial fractures (3).
Impact of Alcohol on Bone Health, Homeostasis, and Fracture Repair
Current Pathobiology Reports, 2020
Purpose of review: Alcohol use continues to rise globally. We review the current literature on the effect of alcohol on bone health, homeostasis and fracture repair to highlight what has been learned in people and animal models of alcohol consumption. Recent findings: Recently, forkhead box O (FoxO) has been found to be upregulated and activated in mesenchymal stem cells (MSC) exposed to alcohol. FoxO has also been found to modulate Wnt/β-catenin signaling, which is necessary for MSC differentiation. Recent evidence suggests alcohol activates FoxO signaling, which may be dysregulating Wnt/β-catenin signaling in MSCs cultured in alcohol. Summary: This review highlights the negative health effects learned from people and chronic and episodic binge alcohol consumption animal models. Studies using chronic alcohol exposure or alcohol exposure then bone fracture repair model have explored several different cellular and molecular signaling pathways important for bone homeostasis and fracture repair, and offer potential for future experiments to explore additional signaling pathways that may be dysregulated by alcohol exposure.
Alcohol and Alcoholism, 2014
Aims: Excessive alcohol consumption is associated with fracture non-union. Canonical Wnt pathway signaling activity regulates normal fracture healing. We previously demonstrated that binge alcohol exposure modulates β-catenin levels in the fracture callus of mice. Here, we sought to determine whether exogenous enhancement β-catenin signaling activity could restore normal fracture healing to binge-exposed mice. Methods: C57BL/6 male mice were exposed to episodic alcohol or saline for 6 total days of alcohol exposure over a 2-week period. Following alcohol exposure, mice were subjected to a stabilized mid-shaft tibia fracture. Beginning 4 days post-injury, mice received daily injections of either lithium chloride or saline subcutaneously. Protein levels of activated, inactivated, and total β-catenin and GSK-3β in fracture calluses were measured at post-injury day 9. Biomechanical strength testing and histology of callus tissue was assessed at post fracture day 14. Results: Binge alcohol was associated with decreased callus biomechanical strength, and reduced cartilaginous callus formation. Alcohol decreased levels of callus-associated activated β-catenin while concomitantly increasing the levels of inactive β-catenin at post-injury day 9. Alcohol also increased callus associated activated GSK-3β at post-injury day 9. Lithium chloride (an inhibitor of GSK-3β) treatment increased activated β-catenin protein levels, significantly decreased activated GSK-3β and restored cartilaginous callus formation and endochondral ossification. Conclusion: These data link alcohol-impaired fracture healing with deregulation of Canonical Wnt signaling activity in the fracture callus. Exogenous activation of the Wnt pathway using LiCl attenuated the damaging effects of binge alcohol exposure on the fracture healing process by modulating canonical Wnt signaling activity.
Alcohol and Alcoholism, 2014
Aims: Excessive alcohol consumption is associated with fracture non-union. Canonical Wnt pathway signaling activity regulates normal fracture healing. We previously demonstrated that binge alcohol exposure modulates β-catenin levels in the fracture callus of mice. Here, we sought to determine whether exogenous enhancement β-catenin signaling activity could restore normal fracture healing to binge-exposed mice. Methods: C57BL/6 male mice were exposed to episodic alcohol or saline for 6 total days of alcohol exposure over a 2-week period. Following alcohol exposure, mice were subjected to a stabilized mid-shaft tibia fracture. Beginning 4 days post-injury, mice received daily injections of either lithium chloride or saline subcutaneously. Protein levels of activated, inactivated, and total β-catenin and GSK-3β in fracture calluses were measured at post-injury day 9. Biomechanical strength testing and histology of callus tissue was assessed at post fracture day 14. Results: Binge alcohol was associated with decreased callus biomechanical strength, and reduced cartilaginous callus formation. Alcohol decreased levels of callus-associated activated β-catenin while concomitantly increasing the levels of inactive β-catenin at post-injury day 9. Alcohol also increased callus associated activated GSK-3β at post-injury day 9. Lithium chloride (an inhibitor of GSK-3β) treatment increased activated β-catenin protein levels, significantly decreased activated GSK-3β and restored cartilaginous callus formation and endochondral ossification. Conclusion: These data link alcohol-impaired fracture healing with deregulation of Canonical Wnt signaling activity in the fracture callus. Exogenous activation of the Wnt pathway using LiCl attenuated the damaging effects of binge alcohol exposure on the fracture healing process by modulating canonical Wnt signaling activity.
Alcoholism: Clinical & Experimental Research, 2004
Background: Alcohol-induced osteoporosis is characterized by a considerable suppression of osteogenesis. The objective of this investigation was to determine the effect of alcohol on gene expression, protein synthesis, and mineralization in human bone marrow-derived mesenchymal stem cells induced toward osteogenic differentiation in vitro. Methods: Human bone marrow-derived mesenchymal stem cells induced toward osteogenesis were cultured in the presence or absence of 50 mM alcohol. Stem cells were characterized by using SH2 antibody to the cell-surface antigen CD105/endoglin, and their proliferation in the presence of alcohol was quantified. The expression of genes for early, middle, and late markers of the osteogenic lineage was quantified by Northern analysis, and bone matrix protein synthesis was assayed. The effect of alcohol on cell-mediated matrix mineralization in terminally differentiated cultures was determined by von Kossa staining. Results: Fluorescence-activated cell sorting analysis of human mesenchymal stem cells separated with a Percoll gradient proved 99% homogeneity by using SH2 antibody to the surface antigen CD105. Dosedependent inhibition of proliferation of these stem cells occurred at concentrations greater than 50 mM alcohol. Gene expression of osteoblast-specific factor 2/core binding factor a1 (Osf2/Cbfa1), type I collagen, alkaline phosphatase, and osteocalcin (early, middle, and late markers for osteogenesis, respectively) was analyzed with and without osteogenic induction and treatment with 50 mM alcohol. After induction, Osf2/Cbfa1 levels were unresponsive to alcohol. To determine the effect of alcohol on human mesenchymal stem cell progression along the osteogenic pathway, messenger RNA (mRNA) levels for type I collagen, alkaline phosphatase, and osteocalcin were examined after osteogenic induction. After osteogenic induction, alcohol down-regulated the gene expression of type I collagen and significantly reduced its synthesis. Alcohol did not alter mRNA expression of alkaline phosphatase, a midstage marker for osteogenesis, but significantly decreased its activity compared with osteogenic induction alone. After induction, osteocalcin remained unchanged by alcohol at both the mRNA and protein levels. Histochemistry revealed decreased alkaline phosphatase staining and fewer alkaline phosphatase-positive cells in alcohol-treated human mesenchymal stem cell cultures. von Kossa staining revealed a reduction in the number of mineralizing nodules in stem cell cultures after alcohol treatment. Conclusions: Collectively, the data suggest that alcohol alters osteogenic differentiation in human bone marrow-derived mesenchymal stem cell cultures during lineage progression and provide further insight into alcohol-induced reduced bone formation.
Alcoholism: Clinical and Experimental Research, 2020
This article is protected by copyright. All rights reserved Background: During bone fracture repair, resident mesenchymal stem cells (MSCs) differentiate into chondrocytes, to form a cartilaginous fracture callus, and osteoblasts, to ossify the collagen matrix. Our laboratory previously reported that alcohol administration led to decreased cartilage formation within the fracture callus of rodents and this effect was mitigated by post-fracture antioxidant treatment. FoxO transcription factors are activated in response to intracellular reactive oxygen species (ROS), and alcohol has been shown to increase ROS. Activation of FoxOs has also been shown to inhibit canonical Wnt signaling, a necessary pathway for MSC differentiation. These findings have led to our hypothesis that alcohol exposure decreases osteochondrogenic differentiation of MSCs through the activation of FoxOs. Methods: Primary rat MSCs were treated with ethanol and assayed for FoxO expression, FoxO activation and downstream target expression. Next, MSCs were differentiated towards osteogenic or chondrogenic lineages in the presence of 50mM ethanol and alterations in osteochondral lineage marker expression was determined. Lastly, osteochondral differentiation experiments were repeated with FoxO1/3 knockdown or with FoxO1/3 inhibitor AS1842856 and osteochondral lineage marker expression was determined. Results: Ethanol increased the expression of FoxO3a at mRNA and protein levels in primary cultured MSCs. This was accompanied by an increase in FoxO1 nuclear localization, FoxO1 activation, and downstream catalase expression. Moreover, ethanol exposure decreased expression of osteogenic and chondrogenic lineage markers. FoxO1/3 knockdown restored pro-osteogenic and pro-chondrogenic lineage marker expression in the presence of 50mM ethanol. However, FoxO1/3 inhibitor only restored pro-osteogenic lineage marker expression. Conclusions: These data show that ethanol has the ability to inhibit MSC differentiation, and this ability may rely, at least partially, on the activation of FoxO transcription factors.