Environmental chemicals termed "obesogens" disrupt the endocrine system to promote adipogenesis and obesity. Tetrabromobisphenol-A (TBBPA) has been reported to increase adipogenesis; however, the mechanism(s) of action are unclear. Thy1 (CD90) is a glycophosphatidylinositol-anchored membrane protein that serves as a marker for stem cells and also plays an important role in regulating adipogenesis and obesity. We investigated whether or not TBBPA promotes adipogenesis in human and mouse cells by reducing Thy1 levels. We further sought to identify the molecular mechanism(s) whereby TBBPA targets Thy1 expression. Mouse and human cells were exposed to TBBPA, and Thy1 expression was analyzed using flow cytometry, Western blotting, and qPCR. We tested whether microRNAs predicted to target Thy1 (miR-103 and miR-107) were upregulated by TBBPA using quantitative PCR assays. We also determined if Thy1 mRNA was a bona fide miR-103/107 target. Our results show that Thy1 expression was reduced in both human and mouse cells after exposure to TBBPA. Both Thy1 mRNA and protein levels were decreased by low-dose TBBPA exposure. TBBPA reduced Thy1 levels and further increased adipogenesis when an adipogenic medium was used. Mechanistically, we show that miR-103 and miR-107 are induced by TBBPA and that miR-103 targets Thy1 to reduce its expression. Our results reveal for the first time that Thy1 is a target of TBBPA. Furthermore, our data support the concept that Thy1 is a key marker targeted by environmental chemicals that promote adipogenesis and obesity.

The obesity epidemic is developing into the most costly health problem facing the world. Obesity, characterized by excessive adipogenesis and enlarged adipocytes, promotes morbidities, such as diabetes, cardiovascular disease, and cancer. Regulation of adipogenesis is critical to our understanding of how fat cell formation causes obesity and associated health problems. Thy1 (also called CD90), a widely used stem cell marker, blocks adipogenesis and reduces lipid accumulation. Thy1‐knockout mice are prone to diet‐induced obesity. Although the importance of Thy1 in adipogenesis and obesity is now evident, how its expression is regulated is not. We hypothesized that DNA methylation has a role in promoting adipogenesis and affects Thy1 expression. Using the methylation inhibitor 5‐aza‐2'‐deoxycytidine (5‐aza‐dC), we investigated whether DNA methylation alters Thy1 expression during adipogenesis in both mouse 3T3‐L1 preadipocytes and mouse mesenchymal stem cells. Thy1 protein and mRNA levels were decreased dramatically during adipogenesis. However, 5‐aza‐dC treatment prevented that phenomenon. Methylation‐sensitive pyrosequencing analysis showed that CpG sites at the Thy1 locus have increased methylation during adipogenesis, as well as increased methylation in adipose tissue from diet‐induced obese mice. These new findings highlight the potential role of Thy1 and DNA methylation in adipogenesis and obesity.—Flores, E. M., Woeller, C. F., Falsetta, M. L., Susiarjo, M., Phipps, R. P. Thy1 (CD90) expression is regulated by DNA methylation during adipogenesis. FASEB J. 33, 3353–3363 (2019). http://www.fasebj.org

Obesity has risen drastically over the last 30 years and occurs through excessive adipogenesis (adipocyte formation) or increases in adipocyte size. Adipocytes produce cytokines such as IL-6 that promotes inflammatory disease. Environmental toxicants termed obesogens such as Tetrabromobisphenol-A (TBBPA) disrupt the endocrine system and increase adipogenesis; however the mechanisms remain unknown. Thy1 (CD90), a cell surface protein, is a member of the immunoglobulin family. We have previously shown that fibroblasts are heterogeneous for Thy1 and only Thy1-/low fibroblasts form adipocytes. We hypothesized that TBBPA promotes adipogenesis by decreasing Thy1 expression. We used mouse 3T3-L1 pre-adipocytes as a model to determine if TBBPA decreases the expression of Thy1 to promote fat cell formation. We measured Thy1 mRNA levels by qPCR and Thy1 protein levels by western blot. Adipogenesis was measured by inspection of lipid droplets and expression of the key adipocyte marker, FABP4. Our data show that TBBPA decreased the expression of Thy1 in 3T3-L1 cells. Furthermore, TBBPA treated cells also expressed higher levels of FABP4, supporting the concept that loss of Thy1 alters cell fate. TBBPA exposure for one week before adipogenic induction resulted in a further reduction of Thy1 and additional increases in FABP4. Thus, prolonged or developmental exposure to TBBPA may affect Thy1 levels and alter physiology long after exposure, which may contribute to the obesity epidemic.

Obesity has been classically linked to poor diet/excessive calorie intake and insufficient physical activity. However, an emerging concept is that environmental pollutants may be driving the global obesity epidemic. The rapid increase in the prevalence of obesity in industrialized, developed and even undeveloped countries cannot be strictly accounted for by poor diet and physical inactivity. Environmental chemicals, termed “obesogens” can promote adipogenesis (fat cell formation). Tetrabromobisphenol‐A (TBBPA), a brominated flame retardant found in infant mattresses and electronics such as cell phones, is an obesogens. However, its mechanism of action remains unknown. We hypothesize that TBBPA promotes adipogenesis by decreasing levels of THY1 (CD90), a cell surface protein present on pre‐adipocytes, certain fibroblasts, and stem cells. We previously determined that fibroblasts are heterogeneous for THY1 expression, where only THY1‐/low fibroblasts differentiate into adipocytes, pointing to THY1's crucial role during adipogenesis and cell fate. Using the well‐established mouse pre‐adipocyte cell line (3T3‐L1), we determined that TBBPA reduces THY1 mRNA and protein levels, promoting adipogenesis. Epigenetic changes, such as increases in global DNA methylation are known to occur during adipogenesis and obesity. DNA hypermethylation typically silences gene expression. Therefore, we hypothesized that obesogens, such as TBBPA, alter epigenetic regulation of THY1 to decrease its expression, thereby priming precursor cells to become adipocytes. Using pyrosequencing, we found that CG rich regions on THY1 are hypermethylated during adipogenesis, resulting in decreased THY1 expression and cell differentiation to adipocytes. Using the methylation inhibitor, 5‐aza‐dC, we determined that the adipogenic effects of TBBPA are blunted when methylation is impeded, which results in increased THY1 levels. Our results confirm THY1 plays a critical role in adipogenesis and that its expression is regulated by increases in DNA methylation. Furthermore, TBBPA, a widely‐used flame retardant, increases DNA methylation at the Thy1 locus to promote adipogenesis. Environmental obesogens contribute to the worldwide obesity epidemic and their further study is critical to combating this epidemic.Support or Funding InformationP30 ES 001247, F31 ES027767‐02This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The obesity epidemic is developing into the most costly health problem facing the world. Obesity, characterized by excessive adipogenesis and enlarged adipocytes, promotes morbidities such as diabetes, cardiovascular disease and cancer. Regulation of adipogenesis is critical to our understanding of how fat cell formation causes obesity and associated health problems. Thy1 (also called CD90), a widely used stem cell marker, blocks adipogenesis and reduces lipid accumulation. Thy1 knockout-mice are prone to diet-induced obesity. While the importance of Thy1 in adipogenesis and obesity is now evident, how its expression is regulated is not. We hypothesized that DNA methylation plays a role in promoting adipogenesis and affects Thy1 expression. Using the methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), we investigated whether DNA methylation alters Thy1 expression during adipogenesis in both mouse 3T3-L1 pre-adipocytes and mouse mesenchymal stem cells. Thy1 protein and mRNA levels were decreased dramatically during adipogenesis. However, 5-aza-dC treatment prevented this phenomenon. Pyrosequencing analysis shows that the CpG sites at the Thy1 locus are methylated during adipogenesis. These new findings highlight the potential role of Thy1 and DNA methylation in adipogenesis and obesity. Results Reduced Thy1 expression during adipogenesis is partially attenuated by 5-aza-dC in bone marrow-derived mouse mesenchymal stem cells.Previously, we demonstrated that pre-adipocytes and mesenchymal stem cells must down-regulate Thy1 expression to differentiate into adipocytes 14 . To determine if DNA methylation regulates Thy1 expression during adipogenesis, we first pharmacologically inhibited DNA methyltransferase in bone-marrow derived mouse Additional InformationCompeting Interests: The authors declare no competing interests.