RHO-Associated Coiled-Coil-Containing Protein Kinase Inhibitors Significantly Modulate the Epithelial–Mesenchymal Transition Induced by TGF-β2 in the 2-D and 3-D Cultures of Human Corneal Stroma Fibroblasts
Background/Objectives: The objective of the present study was to examine the unidentified effects that RHO-associated coiled-coil-containing protein kinase 1 and 2 antagonists exert on the transforming growth factor beta2-induced epithelial–mesenchymal transition of the human corneal stroma. Methods...
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2024-12-01
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| author | Araya Umetsu Yosuke Ida Tatsuya Sato Megumi Higashide Nami Nishikiori Masato Furuhashi Hiroshi Ohguro Megumi Watanabe |
| author_facet | Araya Umetsu Yosuke Ida Tatsuya Sato Megumi Higashide Nami Nishikiori Masato Furuhashi Hiroshi Ohguro Megumi Watanabe |
| author_sort | Araya Umetsu |
| collection | DOAJ |
| description | Background/Objectives: The objective of the present study was to examine the unidentified effects that RHO-associated coiled-coil-containing protein kinase 1 and 2 antagonists exert on the transforming growth factor beta2-induced epithelial–mesenchymal transition of the human corneal stroma. Methods: In the presence or absence of pan-RHO-associated coiled-coil-containing protein kinase inhibitors, ripasudil or Y27632 and RHO-associated coiled-coil-containing protein kinase 2 inhibitor, KD025, we analyzed the following: (1) planar proliferation caused by trans-endothelial electrical resistance and the cellular metabolic characteristics of the two-dimensional cultures of human corneal stroma fibroblasts; (2) the physical properties of a three-dimensional human corneal stroma fibroblasts spheroid; and (3) the gene expressions and their regulators in the extracellular matrix, along with the tissue inhibitors of metalloproteinases and matrix metalloproteinases and the endoplasmic reticulum stress-related factors of the two-dimensional and three-dimensional cultures in human corneal stroma fibroblasts. Results: Exposure to 5 nM of the transforming growth factor beta2 markedly increased the trans-endothelial electrical resistance values as well as the metabolic function in two-dimensional cultures of human corneal stroma fibroblasts. With an increase in stiffening, this exposure also reduced the size of three-dimensional human corneal stroma fibroblast spheroids, which are typical cellular phenotypes of the epithelial–mesenchymal transition. Both pan-RHO-associated coiled-coil-containing protein kinase inhibitors and RHO-associated coiled-coil-containing protein kinase 2 inhibitors substantially modulated these transforming growth factor beta2-induced effects, albeit in a different manner. Gene expression analysis supported such biological alterations via either with transforming growth factor beta2 alone or with the RHO-associated coiled-coil-containing protein kinase inhibitors variants with the noted exception being the transforming growth factor beta2-induced effects toward the three-dimensional human corneal stroma fibroblast spheroid. Conclusions: The findings presented herein suggest the following: (1) the epithelial–mesenchymal transition could be spontaneously evoked in the three-dimensional human corneal stroma fibroblast spheroid, and, therefore, the epithelial–mesenchymal transition induced by transforming growth factor beta2 could differ between two-dimensional and three-dimensional cultured HCSF cells; and (2) the inhibition of ROCK1 and 2 significantly modulates the transforming growth factor beta2-induced an epithelial–mesenchymal transition in both two-dimensionally and three-dimensionally cultured human corneal stroma fibroblasts, albeit in a different manner. |
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| publishDate | 2024-12-01 |
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| spelling | doaj-art-bd2b9362fb224762aa81b8aa2bd724ce2025-08-20T02:57:12ZengMDPI AGBiomedicines2227-90592024-12-011212278410.3390/biomedicines12122784RHO-Associated Coiled-Coil-Containing Protein Kinase Inhibitors Significantly Modulate the Epithelial–Mesenchymal Transition Induced by TGF-β2 in the 2-D and 3-D Cultures of Human Corneal Stroma FibroblastsAraya Umetsu0Yosuke Ida1Tatsuya Sato2Megumi Higashide3Nami Nishikiori4Masato Furuhashi5Hiroshi Ohguro6Megumi Watanabe7Department of Ophthalmology, School of Medicine, Sapporo Medical University, S1 W16 Chuo-ku, Sapporo City 060-8543, Hokkaido, JapanDepartment of Ophthalmology, School of Medicine, Sapporo Medical University, S1 W16 Chuo-ku, Sapporo City 060-8543, Hokkaido, JapanDepartment of Cardiovascular, Renal and Metabolic Medicine, School of Medicine, Sapporo Medical University, S1 W16 Chuo-ku, Sapporo City 060-8543, Hokkaido, JapanDepartment of Ophthalmology, School of Medicine, Sapporo Medical University, S1 W16 Chuo-ku, Sapporo City 060-8543, Hokkaido, JapanDepartment of Ophthalmology, School of Medicine, Sapporo Medical University, S1 W16 Chuo-ku, Sapporo City 060-8543, Hokkaido, JapanDepartment of Cardiovascular, Renal and Metabolic Medicine, School of Medicine, Sapporo Medical University, S1 W16 Chuo-ku, Sapporo City 060-8543, Hokkaido, JapanDepartment of Ophthalmology, School of Medicine, Sapporo Medical University, S1 W16 Chuo-ku, Sapporo City 060-8543, Hokkaido, JapanDepartment of Ophthalmology, School of Medicine, Sapporo Medical University, S1 W16 Chuo-ku, Sapporo City 060-8543, Hokkaido, JapanBackground/Objectives: The objective of the present study was to examine the unidentified effects that RHO-associated coiled-coil-containing protein kinase 1 and 2 antagonists exert on the transforming growth factor beta2-induced epithelial–mesenchymal transition of the human corneal stroma. Methods: In the presence or absence of pan-RHO-associated coiled-coil-containing protein kinase inhibitors, ripasudil or Y27632 and RHO-associated coiled-coil-containing protein kinase 2 inhibitor, KD025, we analyzed the following: (1) planar proliferation caused by trans-endothelial electrical resistance and the cellular metabolic characteristics of the two-dimensional cultures of human corneal stroma fibroblasts; (2) the physical properties of a three-dimensional human corneal stroma fibroblasts spheroid; and (3) the gene expressions and their regulators in the extracellular matrix, along with the tissue inhibitors of metalloproteinases and matrix metalloproteinases and the endoplasmic reticulum stress-related factors of the two-dimensional and three-dimensional cultures in human corneal stroma fibroblasts. Results: Exposure to 5 nM of the transforming growth factor beta2 markedly increased the trans-endothelial electrical resistance values as well as the metabolic function in two-dimensional cultures of human corneal stroma fibroblasts. With an increase in stiffening, this exposure also reduced the size of three-dimensional human corneal stroma fibroblast spheroids, which are typical cellular phenotypes of the epithelial–mesenchymal transition. Both pan-RHO-associated coiled-coil-containing protein kinase inhibitors and RHO-associated coiled-coil-containing protein kinase 2 inhibitors substantially modulated these transforming growth factor beta2-induced effects, albeit in a different manner. Gene expression analysis supported such biological alterations via either with transforming growth factor beta2 alone or with the RHO-associated coiled-coil-containing protein kinase inhibitors variants with the noted exception being the transforming growth factor beta2-induced effects toward the three-dimensional human corneal stroma fibroblast spheroid. Conclusions: The findings presented herein suggest the following: (1) the epithelial–mesenchymal transition could be spontaneously evoked in the three-dimensional human corneal stroma fibroblast spheroid, and, therefore, the epithelial–mesenchymal transition induced by transforming growth factor beta2 could differ between two-dimensional and three-dimensional cultured HCSF cells; and (2) the inhibition of ROCK1 and 2 significantly modulates the transforming growth factor beta2-induced an epithelial–mesenchymal transition in both two-dimensionally and three-dimensionally cultured human corneal stroma fibroblasts, albeit in a different manner.https://www.mdpi.com/2227-9059/12/12/2784three-dimensional spheroid cultureTGF-β2human corneal stroma fibroblasts (HCSFs)corneal injury modelROCK inhibitor |
| spellingShingle | Araya Umetsu Yosuke Ida Tatsuya Sato Megumi Higashide Nami Nishikiori Masato Furuhashi Hiroshi Ohguro Megumi Watanabe RHO-Associated Coiled-Coil-Containing Protein Kinase Inhibitors Significantly Modulate the Epithelial–Mesenchymal Transition Induced by TGF-β2 in the 2-D and 3-D Cultures of Human Corneal Stroma Fibroblasts Biomedicines three-dimensional spheroid culture TGF-β2 human corneal stroma fibroblasts (HCSFs) corneal injury model ROCK inhibitor |
| title | RHO-Associated Coiled-Coil-Containing Protein Kinase Inhibitors Significantly Modulate the Epithelial–Mesenchymal Transition Induced by TGF-β2 in the 2-D and 3-D Cultures of Human Corneal Stroma Fibroblasts |
| title_full | RHO-Associated Coiled-Coil-Containing Protein Kinase Inhibitors Significantly Modulate the Epithelial–Mesenchymal Transition Induced by TGF-β2 in the 2-D and 3-D Cultures of Human Corneal Stroma Fibroblasts |
| title_fullStr | RHO-Associated Coiled-Coil-Containing Protein Kinase Inhibitors Significantly Modulate the Epithelial–Mesenchymal Transition Induced by TGF-β2 in the 2-D and 3-D Cultures of Human Corneal Stroma Fibroblasts |
| title_full_unstemmed | RHO-Associated Coiled-Coil-Containing Protein Kinase Inhibitors Significantly Modulate the Epithelial–Mesenchymal Transition Induced by TGF-β2 in the 2-D and 3-D Cultures of Human Corneal Stroma Fibroblasts |
| title_short | RHO-Associated Coiled-Coil-Containing Protein Kinase Inhibitors Significantly Modulate the Epithelial–Mesenchymal Transition Induced by TGF-β2 in the 2-D and 3-D Cultures of Human Corneal Stroma Fibroblasts |
| title_sort | rho associated coiled coil containing protein kinase inhibitors significantly modulate the epithelial mesenchymal transition induced by tgf β2 in the 2 d and 3 d cultures of human corneal stroma fibroblasts |
| topic | three-dimensional spheroid culture TGF-β2 human corneal stroma fibroblasts (HCSFs) corneal injury model ROCK inhibitor |
| url | https://www.mdpi.com/2227-9059/12/12/2784 |
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