STAT3 Is the Master Regulator for the Forming of 3D Spheroids of 3T3-L1 Preadipocytes

Abstract

To elucidate the currently unknown mechanisms responsible for the diverse biological aspects between two-dimensional (2D) and three-dimensional (3D) cultured 3T3-L1 preadipocytes, RNA-sequencing analyses were performed. During a 7-day culture period, 2D- and 3D-cultured 3T3-L1 cells were subjected to lipid staining by BODIPY, qPCR for adipogenesis related genes, including peroxisome proliferator-activated receptor γ (Pparγ), CCAAT/enhancer-binding protein alpha (Cebpa), Ap2 (fatty acid-binding protein 4; Fabp4), leptin, and AdipoQ (adiponectin), and RNA-sequencing analysis. Differentially expressed genes (DEGs) were detected by next-generation RNA sequencing (RNA-seq) and validated by a quantitative reverse transcription–polymerase chain reaction (qRT–PCR). Bioinformatic analyses were performed on DEGs using a Gene Ontology (GO) enrichment analysis and an Ingenuity Pathway Analysis (IPA). Significant spontaneous adipogenesis was observed in 3D 3T3-L1 spheroids, but not in 2D-cultured cells. The mRNA expression of Pparγ, Cebpa, and Ap2 among the five genes tested were significantly higher in 3D spheroids than in 2D-cultured cells, thus providing support for this conclusion. RNA analysis demonstrated that a total of 826 upregulated and 725 downregulated genes were identified as DEGs. GO enrichment analysis and IPA found 50 possible upstream regulators, and among these, 6 regulators—transforming growth factor β1 (TGFβ1), signal transducer and activator of transcription 3 (STAT3), interleukin 6 (IL6), angiotensinogen (AGT), FOS, and MYC—were, in fact, significantly upregulated. Further analyses of these regulators by causal networks of the top 14 predicted diseases and functions networks (IPA network score indicated more than 30), suggesting that STAT3 was the most critical upstream regulator. The findings presented herein suggest that STAT3 has a critical role in regulating the unique biological properties of 3D spheroids that are produced from 3T3-L1 preadipocytes.

Hypoxia Differently Affects TGF-β2-Induced Epithelial Mesenchymal Transitions in the 2D and 3D Culture of the Human Retinal Pigment Epithelium Cells

“…In their subsequent study, they also demonstrated that TGF-β2 stimulated EMT in RPE, caused the significant down-regulation of PGC1α, and mitochondrial dysfunctions as well as a metabolic shift towards reduced OXPHOS and increased glycolysis [ 16 ]. In the current study, although we also found that TGF-β2 and hypoxia synergistically and differently induced EMT of the HRPE cells considering the fluctuation in the mRNA expression of HIF1α and the mitochondrial metabolism described above, those fluctuations were also different between 2D and 3D cultures, and such diversity between them may be caused by possible up-stream regulators requiring the generation of the 3D spheroids, as was recently determined using 3T3-L1 cells [ 36 ].…”

“…To study further in terms of the difference between 2D and 3D HRPE cells under several conditions as above, qPCR analysis of the recently determined possible up-stream regulators forming 3D spheroids; STAT3 , IL6 , FOS , TGFb1 , AGT and MYC using 3T3-L1 cells [ 36 ] were studied. As shown in Figure 9 , among these regulators, mRNA expressions of STAT3 , IL6 , FOS and TGF-β1 of the 3D HRPE spheroids were substantially higher than those of the 2D HRPE cells.…”

“… Effects of TGF-β2 on mRNA expression of the possible up-stream regulators of 3D spheroids; STAT3 , IL6 , FOS , TGFb1 , AGT and MYC of 2D and 3D HRPE cells under normoxia or hypoxia conditions. Two-dimensional and three-dimensional cultured HRPE cells at Day 6 prepared under normoxia or hypoxia conditions were treated without (control) or with a 5 ng/mL solution of TGF-β2 (TGFβ), and each sample was subjected to qPCR analysis and the expression of mRNA in the possible up-stream regulators requiring for the generation of the 3D spheroids [ 36 ]; STAT3 , IL6 , FOS , TGFb1 , AGT and MYC , were estimated. All experiments were performed in duplicate using fresh preparations (2D; n = 5, 3D; n = 10 spheroids each).…”

Brimonidine Modulates the ROCK1 Signaling Effects on Adipogenic Differentiation in 2D and 3D 3T3-L1 Cells

“…To identify possible mechanisms responsible for causing such a difference between 2D and 3D cell cultures, RNA sequence analyses were performed. The results suggested that STAT3 functions as the master regulator in forming the 3D spheroid architecture, and related signaling may induce these unique biological differences [ 52 ]. However, as of this writing, the relationship between DIF+ and STAT3-related signaling, as well as diversity in the nature of orbital and other systemic adipocytes, has not been fully investigated.…”

All-trans Retinoic Acids Synergistically and Beneficially Affect In Vitro Glaucomatous Trabecular Meshwork (TM) Models Using 2D and 3D Cell Cultures of Human TM Cells

“…As a real-time cellular metabolic function analysis, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of the 2D-cultured HTM cells in the absence or presence of TGF-β2 (5 ng/mL) and/or ATRA (10 μM) were measured using a Seahorse XFe96 Bioanalyzer (Agilent Technologies, Santa Clara, CA, U.S.A.), as described in a recent report [ 43 , 44 , 45 ]. Briefly, 20 × 10 3 2D HTM cells under several conditions—(1) nontreated control, (2) treated with TGF-β2, (3) treated with ATRA, and (4) treated with TGF-β2 and ATRA—were grown in 96-well assay plates.…”