G-Protein-Coupled Receptors Mediate Modulations of Cell Viability and Drug Sensitivity by Aberrantly Expressed Recoverin 3 within A549 Cells.

To elucidate the currently unknown molecular mechanisms responsible for the aberrant expression of recoverin (Rec) within cancerous cells, we examined two-dimensional (2D) and three-dimensional (3D) cultures of Rec-negative lung adenocarcinoma A549 cells which had been transfected with a plasmid containing human recoverin cDNA (A549 Rec) or an empty plasmid as a mock control (A549 MOCK). Using these cells, we measured cytotoxicity by several anti-tumor agents (2D), cellular metabolism including mitochondrial and glycolytic functions by a Seahorse bio-analyzer (2D), the physical properties, size and stiffness of the 3D spheroids, trypsin sensitivities (2D and 3D), and RNA sequencing analysis (2D). Compared with the A549 MOCK, the A549 Rec cells showed (1) more sensitivity toward anti-tumor agents (2D) and a 0.25% solution of trypsin (3D); (2) a metabolic shift from glycolysis to oxidative phosphorylation; and (3) the formation of larger and stiffer 3D spheroids. RNA sequencing analysis and bioinformatic analyses of the differentially expressed genes (DEGs) using Gene Ontology (GO) enrichment analysis suggested that aberrantly expressed Rec is most likely associated with several canonical pathways including G-protein-coupled receptor (GPCR)-mediated signaling and signaling by the cAMP response element binding protein (CREB). The findings reported here indicate that the aberrantly expressed Rec-induced modulation of the cell viability and drug sensitivity may be GPCR mediated.

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

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 ß1 (TGFß1), 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.

Addition of ROCK inhibitors to prostaglandin derivative (PG) synergistically affects adipogenesis of the 3D spheroids of human orbital fibroblasts (HOFs).

To study the additive effects of Rho-associated coiled-coil containing protein kinase inhibitors, ripasudil (Rip) to bimatoprost acid (BIM-A) on orbital adipose tissue, three-dimensional (3D) cultures of human orbital fibroblasts (HOFs) were prepared and the physical properties including the 3D spheroid size and stiffness, lipid staining by BODIPY and the mRNA expression of adipogenesis-related genes, PPARγ and AP2, and extracellular matrix (ECM) including collagen (COL)1, 4 and 6, and fibronectin (FN) were analyzed. Adipogenesis (DIF+) induced (1) enlargement and increasing stiffness of the 3D HOFs spheroid, (2) increased lipid staining, the expression of adipogenesis-related gene expressions, and (3) the down-regulation of COL1 and FN and up-regulation of COL4 and COL6. In the presence of BIM-A, (1) such DIF+-induced changes in 3D spheroid size and stiffness were significantly inhibited or enhanced, respectively, (2) the lipid staining and its related gene expressions were significantly down-regulated, and (3) the expression of COL1 and COL6 were up-regulated. By the addition of Rip to BIM-A, the above BIM-A-induced effects were all inhibited, except for the up-regulation of COL6 and FN expression, that is, enlarging and decreasing stiffness, enhancement of lipid staining and its related gene expression, and down-regulation of COL1 expression. Our present study indicates that Rip significantly suppressed BIM-A-induced effects toward 3D HOFs spheroids.

Reactivities of a Prostanoid EP2 Agonist, Omidenepag, Are Useful for Distinguishing between 3D Spheroids of Human Orbital Fibroblasts without or with Graves' Orbitopathy.

BACKGROUND: To obtain new insights into the activation of the thyroid-stimulating hormone (TSH) and insulin-like growth factor 1 (IGF-1) receptors in human orbital fibroblasts (n-HOFs), the effects of the prostanoid EP2 agonist, omidenepag (OMD), and a rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) were evaluated using three-dimension (3D) n-HOFs spheroids in the absence and presence of the recombinant human TSH receptor antibodies, M22 and IGF-1. METHODS: The effects of 100 nM OMD or 10 µM Rip on the physical properties, size, stiffness, and mRNA expression of several extracellular matrix (ECM) molecules, their regulator, inflammatory cytokines, and endoplasmic reticulum (ER) stress-related factors were examined and compared among 3D spheroids of n-HOFs, M22-/IGF-1-activated n-HOFs and GO-related human orbital fibroblasts (GHOFs). RESULTS: The physical properties and mRNA expressions of several genes of the 3D n-HOFs spheroids were significantly and diversely modulated by the presence of OMD or Rip. The OMD-induced effects on M22-/IGF-1-activated n-HOFs were similar to the effects caused by GHOHs, but quite different from those of n-HOFs. CONCLUSIONS: The findings presented herein indicate that the changes induced by OMD may be useful in distinguishing between n-HOFs and GHOFs.

ROCK inhibitors modulate the physical properties and adipogenesis of 3D spheroids of human orbital fibroblasts in different manners.

To elucidate the pharmacological effects of Rho-associated coiled-coil containing protein kinase inhibitors (ROCK-is), ripasudil (Rip), Y27632, and KD025, on human orbital fatty tissue, the human orbital fibroblasts (HOFs) were three-dimensional (3D) cultured for 12 days. The effects of ROCK-is on the physical properties of the 3D-cultured HOF spheroids, including their sizes and physical stiffness, their adipogenesis by lipid staining, and the mRNA expression of adipogenesis-related genes, PPARγ and AP2, and extracellular matrix (ECM) including collagen (COL) 1, 4, and 6, and fibronectin were analyzed. A significant increase in the sizes, physical stiffness, lipid staining, and mRNA expression of adipogenesis-related genes, COL4 and COL6, and a decrease in COL1 expression were observed with adipogenesis (DIF+). In the presence of ROCK-is, such DIF+-induced effects were differently modulated as follows: (1) the sizes were not affected or significantly enhanced by Rip, Y27632, or KD025, (2) the physical stiffness was significantly decreased in Rip and Y27632, but was substantially increased in KD025, (3) the lipid staining was further enhanced or significantly suppressed by Rip, Y27632, or KD025, and both PPARγ and AP2 expression were significantly downregulated or upregulated by KD025 or Rip, and (4) Rip upregulated the expression of COL4, Y27632 upregulated the expression of COL1, COL4, and COL6, and KD025 upregulated the expression of COL1 and COL4. This study indicates that ROCK-is significantly and differently modulate physical properties of the 3D HOF spheroids as well as their adipogenesis.

Prostaglandin F2α and EP2 agonists, and a ROCK inhibitor modulate the formation of 3D organoids of Grave's orbitopathy related human orbital fibroblasts.

3D organoid cultures were used to elucidate the periocular effects of several anti-glaucoma drugs including a prostaglandin F2α analogue (bimatoprost acid; BIM-A), EP2 agonist (omidenepag; OMD) or a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor (ripasudil; Rip) on Grave's orbitopathy (GO) related orbital fatty tissue. 3D organoids were prepared from GO related human orbital fibroblasts (GHOFs) obtained from patients with GO. The effects of either 100 nM BIM-A, 100 nM OMD or 10 µM Rip on the 3D GHOFs organoids were examined with respect to organoid size, physical properties by a micro-squeezer, and the mRNA expression of extracellular matrix (ECM) proteins including collagen (COL) 1, COL 4, COL 6, and fibronectin (FN), ECM regulatory genes including lysyl oxidase (LOX), Connective Tissue Growth Factor (CTGF) and inflammatory cytokines including interleukin-1ß (IL1ß) and interleukin-6 (IL6). The size of the 3D GHOFs organoids decreased substantially in the presence of BIM-A, but also increased substantially in the presence of the others (OMD or Rip). The physical stiffness of the 3D GHOFs organoids was significantly decreased by Rip. BIM-A caused significantly the down-regulation of three ECM genes, Col 1, Col 6 and Fn, and two ECM regulatory genes and the up-regulation of IL6. In the presence of OMD, two ECM genes, Col 1 and Fn, and LOX were significantly down-regulated but IL1ß and IL6 were significantly up-regulated. In the case of Rip, Col 1, FN and CTGF were significant down-regulated. Our present findings indicate that anti-glaucoma drugs modulate the structures and physical properties 3D GHOFs organoids in different manners by modifying the gene expressions of ECM, ECM regulatory factors and inflammatory cytokines. The results indicate that the benefits and demerits of anti-glaucoma medications need to be scrutinized carefully, in cases of patients with GO.