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AMF30a Promotes Survival and Function of Human Corneal Endothelial Cells by Regulating TGF-β/ROCK/HIPPO Pathway

ABSTRACT: Purpose: Corneal endothelial cells (CECs), located in the innermost layer of cornea, are crucial for maintaining its transparency. Peptidylarginine deiminase 2 (PAD2) is an enzyme responsible for catalyzing the post-translational modification of arginine into citrulline, a process known as citrullination. In this study, we investigated the effect of AMF30a, PAD2 inhibitor, on survival and function of CECs. Methods: Cultured human CECs (hCECs) were treated with AMF30a, followed by treatments with or without AMF30a under transforming growth factor-beta (TGF-β). Cells were cultured from donor corneas and analyzed for viability (CCK-8), proliferation (BrdU assay), cytotoxicity (LDH assay), and oxidative stress (DCF-DA). Adhesion and morphology were evaluated via crystal violet staining and imaging software. Western blot and immunofluorescence identified protein expression and localization. RNA sequencing revealed differentially expressed genes, with functional enrichment via GO analysis. Results: AMF30a enhanced cell viability, proliferation, and adhesion while reducing cytotoxicity and oxidative stress. Morphological analysis revealed reduced cell size and elongation factor, indicating structural changes. AMF30a modulated the HIPPO signaling pathway by increasing YAP phosphorylation and reducing its nuclear translocation, while downregulating ERK1/2 activation. Transcriptome analysis identified differentially expressed genes (DEGs) associated with AMF30a treatment, highlighting pathways related to GPCR signaling and protein targeting. Additionally, AMF30a counteracted TGF-β-induced effects, including increased oxidative stress, citrullination, and ROCK/HIPPO signaling activation, thereby promoting cell cycle progression and reducing senescence. Conclusion: PAD2-mediated citrullination is essential for TGF-β/ROCK/HIPPO signaling pathway. AMF30a promoted the proliferation and protected against TGF-β-induced senescence in hCECs, suggesting that PAD2 plays a significant role in the survival and function of hCECs. Thus, AMF30a may be a promising therapeutic strategy for hCEC diseases.

ORGANISM(S): Homo sapiens

PROVIDER: GSE289332 | GEO | 2025/08/06

REPOSITORIES: GEO

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	GSE289332_Expression_Profile.GRCh38.gene.txt.gz	Txt

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Project description:Important remodeling of the extracellular matrix is a hallmark of corneal wound healing. Besides the massive secretion of fibronectin (FN) that characterizes the early step of that process, alterations in the secretion of collagens also occurs as part of this wound healingresponse. In this study, we examined whether expression of the gene encoding the M-NM-15 subunit from the FN binding integrin M-NM-15M-NM-21 changes as corneal epithelial cells (CECs) are cultured in the presence of collagens. Responsiveness of the M-NM-15 gene toward collagen was determined by transfection of recombinant plasmids bearing the CAT reporter gene under the control of various segments from the human M-NM-15 promoter into primary cultured rabbit (RCECs) and human (HCECs) CECs cultured on BSA or on collagens. Electrophoretic mobility shift assays (EMSAs) and Western blot analyses were used to monitor both the DNA binding and expression of the transcription factors required to ensure basal transcription of the M-NM-15 gene. The influence collagens on the patterns of genes expressed by HCECs was also studied be gene profiling on microarrays. All collagen types repressed the transcriptional activity directed by the full-length M-NM-15 promoter/CAT construct in confluent CECs. A moderate increase in M-NM-15 promoter activity was observed in subconfluent RCECs grown on CIV but not on CI. These collagen-dependent regulatory influences also correlated with alterations in either the expression or the DNA binding of the transcription factors Sp1/Sp3, NFI and AP-1 that ensure basal transcription of the M-NM-15 gene. Gene profiling on microarray revealed that CI more profoundly alter the pattern of genes expressed by HCECs than what is observed with CIV. Collagens considerably suppressed expression of the M-NM-15 gene in CECs at confluence suggesting that the sustained staining for CI and CIV after corneal injury may play a role in controlling adhesion to the temporary FN matrix and further differentiation of CECs into suprabasal epithelial cells through a mechanism involving the M-NM-15M-NM-21 integrin. Primary cultures of human corneal epithelial cells cultivated on BSA (number of replicates: 2), Collagen type I (number of replicates: 2) and Collagen type IV (number of replicates: 2) matrix.

Project description:Abstract: Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. It is well established that a gonadotropin releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope derived LBT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterize the temporal dynamics of this modification. Our results show that actin and tubulin are rapidly citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. Data shows that 10 nM GnRHa induces citrullination of beta-actin with maximal levels occurring at 10 minutes. The level of beta-actin citrullination is reduced in the presence of the pan-PAD inhibitor bi-phenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa induced actin reorganization in dispersed mouse gonadotrope cells. GnRHa induces citrullination of beta-tubulin with elevated levels occurring at 30 minutes; similarly to beta-actin, this response is attenuated in the presence of BB-ClA. To examine the functional consequence of beta-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LBT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that MT average lifetime increases following 30 minutes of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells. Keywords: gonadotrope, peptidylarginine deiminase, citrullination, cytoskeleton, beta tubulin, beta actin, microtubules

Project description:Important remodeling of the extracellular matrix is a hallmark of corneal wound healing. Besides the massive secretion of fibronectin (FN) that characterizes the early step of that process, alterations in the secretion of collagens also occurs as part of this wound healingresponse. In this study, we examined whether expression of the gene encoding the α5 subunit from the FN binding integrin α5β1 changes as corneal epithelial cells (CECs) are cultured in the presence of collagens. Responsiveness of the α5 gene toward collagen was determined by transfection of recombinant plasmids bearing the CAT reporter gene under the control of various segments from the human α5 promoter into primary cultured rabbit (RCECs) and human (HCECs) CECs cultured on BSA or on collagens. Electrophoretic mobility shift assays (EMSAs) and Western blot analyses were used to monitor both the DNA binding and expression of the transcription factors required to ensure basal transcription of the α5 gene. The influence collagens on the patterns of genes expressed by HCECs was also studied be gene profiling on microarrays. All collagen types repressed the transcriptional activity directed by the full-length α5 promoter/CAT construct in confluent CECs. A moderate increase in α5 promoter activity was observed in subconfluent RCECs grown on CIV but not on CI. These collagen-dependent regulatory influences also correlated with alterations in either the expression or the DNA binding of the transcription factors Sp1/Sp3, NFI and AP-1 that ensure basal transcription of the α5 gene. Gene profiling on microarray revealed that CI more profoundly alter the pattern of genes expressed by HCECs than what is observed with CIV. Collagens considerably suppressed expression of the α5 gene in CECs at confluence suggesting that the sustained staining for CI and CIV after corneal injury may play a role in controlling adhesion to the temporary FN matrix and further differentiation of CECs into suprabasal epithelial cells through a mechanism involving the α5β1 integrin.

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AMF30a Promotes Survival and Function of Human Corneal Endothelial Cells by Regulating TGF-β/ROCK/HIPPO Pathway

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