{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,26]],"date-time":"2026-07-26T20:46:31Z","timestamp":1785098791206,"version":"3.55.0"},"reference-count":54,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2020,12,26]],"date-time":"2020-12-26T00:00:00Z","timestamp":1608940800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JCM"],"abstract":"<jats:p>The lamina cribrosa (LC) is a key site of fibrotic damage in glaucomatous optic neuropathy and the precise mechanisms of LC change remain unclear. Elevated Ca2+ is a major driver of fibrosis, and therefore intracellular Ca2+ signaling pathways are relevant glaucoma-related mechanisms that need to be studied. Protein kinase C (PKC), mitogen-activated MAPK kinases (p38 and p42\/44-MAPK), and the PI3K\/mTOR axis are key Ca2+ signal transducers in fibrosis and we therefore investigated their expression and activity in normal and glaucoma cultured LC cells. We show, using Western immune-blotting, that hyposmotic-induced cellular swelling activates PKC\u03b1, p42\/p44, and p38 MAPKs, the activity is transient and biphasic as it peaks between 2 min and 10 min. The expression and activity of PKC\u03b1, p38 and p42\/p44-MAPKs are significantly (p &lt; 0.05) increased in glaucoma LC cells at basal level, and at different time-points after hyposmotic stretch. We also found elevated mRNA expression of mRNA expression of PI3K, IP3R, mTOR, and CaMKII in glaucoma LC cells. This study has identified abnormalities in multiple calcium signaling pathways (PKC\u03b1, MAPK, PI3K) in glaucoma LC cells, which might have significant functional and therapeutic implications in optic nerve head (ONH) fibrosis and cupping in glaucoma.<\/jats:p>","DOI":"10.3390\/jcm10010062","type":"journal-article","created":{"date-parts":[[2020,12,27]],"date-time":"2020-12-27T20:04:58Z","timestamp":1609099498000},"page":"62","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Intra-Cellular Calcium Signaling Pathways (PKC, RAS\/RAF\/MAPK, PI3K) in Lamina Cribrosa Cells in Glaucoma"],"prefix":"10.3390","volume":"10","author":[{"given":"Mustapha","family":"Irnaten","sequence":"first","affiliation":[{"name":"Department Ophthalmology, Mater Misericordiae University Hospital, D07 R2WY Dublin, Ireland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aisling","family":"Duff","sequence":"additional","affiliation":[{"name":"Milton Medical Centre New South Wales, Milton, NSW 2538, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3594-6560","authenticated-orcid":false,"given":"Abbot","family":"Clark","sequence":"additional","affiliation":[{"name":"Department Pharmacology &amp; Neuroscience and the North Texas Eye Research Institute, Health Science Center, Fort Worth, TX 76107, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Colm","family":"O\u2019Brien","sequence":"additional","affiliation":[{"name":"Department Ophthalmology, Mater Misericordiae University Hospital, D07 R2WY Dublin, Ireland"},{"name":"School of Medicine and Medical Science, University College Dublin, D04 V1W8 Dublin, Ireland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,12,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1136\/bjo.2005.081224","article-title":"The number of people with glaucoma worldwide in 2010 and 2020","volume":"90","author":"Quigley","year":"2006","journal-title":"Br. J. Ophthalmol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1016\/0002-9394(83)90389-6","article-title":"Morphologic Changes in the Lamina Cribrosa Correlated with Neural Loss in Open-Angle Glaucoma","volume":"95","author":"Quigley","year":"1983","journal-title":"Am. J. Ophthalmol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1016\/S0002-9394(14)75984-7","article-title":"Changes in the Extracellular Matrix of the Human Optic Nerve Head in Primary Open-Angle Glaucoma","volume":"109","author":"Hernandez","year":"1990","journal-title":"Am. J. Ophthalmol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1006\/exer.1994.1079","article-title":"Collagen Type IV Gene Expression in Human Optic Nerve Heads with Primary Open Angle Glaucoma","volume":"59","author":"Hernandez","year":"1994","journal-title":"Exp. Eye Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"552","DOI":"10.1136\/bjo.75.9.552","article-title":"Alterations in elastin of the optic nerve head in human and experimental glaucoma","volume":"75","author":"Quigley","year":"1991","journal-title":"Br. J. Ophthalmol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1136\/bjo.83.2.209","article-title":"Transforming growth factor beta isoforms in human optic nerve heads","volume":"83","author":"Pena","year":"1999","journal-title":"Br. J. Ophthalmol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"896","DOI":"10.1001\/jama.2013.1363","article-title":"Association of Fibrosis With Mortality and Sudden Cardiac Death in Patients With Nonischemic Dilated Cardiomyopathy","volume":"309","author":"Gulati","year":"2013","journal-title":"JAMA"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"795","DOI":"10.1183\/09031936.00185114","article-title":"Global incidence and mortality of idiopathic pulmonary fibrosis: A systematic review","volume":"46","author":"Hutchinson","year":"2015","journal-title":"Eur. Respir. J."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1138","DOI":"10.1056\/NEJMra1300575","article-title":"Fibrosis\u2014A Common Pathway to Organ Injury and Failure","volume":"372","author":"Rockey","year":"2015","journal-title":"N. Engl. J. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1002\/path.2277","article-title":"Cellular and molecular mechanisms of fibrosis","volume":"214","author":"Wynn","year":"2007","journal-title":"J. Pathol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"576","DOI":"10.1172\/JCI31030","article-title":"Fibrosis and diseases of the eye","volume":"117","author":"Friedl","year":"2007","journal-title":"J. Clin. Investig."},{"key":"ref_12","first-page":"283","article-title":"SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer","volume":"510","author":"Mazur","year":"2014","journal-title":"Nat. Cell Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1186\/s13578-018-0223-5","article-title":"A temporal examination of calcium signaling in cancer- from tumorigenesis, to immune evasion, and metastasis","volume":"8","author":"Xu","year":"2018","journal-title":"Cell Biosci."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"S14","DOI":"10.1097\/00005344-198710001-00004","article-title":"Structure and Function of Calcium-Binding Proteins","volume":"10","author":"Hiraoki","year":"1987","journal-title":"J. Cardiovasc. Pharmacol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1016\/bs.pmbts.2015.06.007","article-title":"Intraocular Pressure and the Mechanisms Involved in Resistance of the Aqueous Humor Flow in the Trabecular Meshwork Outflow Pathways","volume":"134","author":"Tamm","year":"2015","journal-title":"Prog. Mol. Biol. Transl. Sci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"C611","DOI":"10.1152\/ajpcell.00054.2020","article-title":"The role of lamina cribrosa tissue stiffness and fibrosis as fundamental biomechanical drivers of pathological glaucoma cupping","volume":"319","author":"Hopkins","year":"2020","journal-title":"Am. J. Physiol. Physiol."},{"key":"ref_17","first-page":"76","article-title":"Differential global and extra-cellular matrix focused gene expression patterns between normal and glau-comatous human lamina cribrosa cells","volume":"15","author":"Kirwan","year":"2009","journal-title":"Mol. Vis."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1002\/glia.20247","article-title":"Transforming growth factor-\u03b2-regulated gene transcription and protein expression in human GFAP-negative lamina cribrosa cells","volume":"52","author":"Kirwan","year":"2005","journal-title":"Glia"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1097\/00061198-200408000-00011","article-title":"Effect of Cyclical Mechanical Stretch and Exogenous Transforming Growth Factor-\u03b21 on Matrix Metalloproteinase-2 Activity in Lamina Cribrosa Cells from the Human Optic Nerve Head","volume":"13","author":"Kirwan","year":"2004","journal-title":"J. Glaucoma"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2243","DOI":"10.1167\/iovs.11-6729","article-title":"Hypoxia Regulated Gene Transcription in Human Optic Nerve Lamina Cribrosa Cells in Culture","volume":"53","author":"Kirwan","year":"2012","journal-title":"Investig. Opthalmol. Vis. Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/j.survophthal.2017.06.007","article-title":"Aging and ocular tissue stiffness in glaucoma","volume":"63","author":"Liu","year":"2018","journal-title":"Surv. Ophthalmol."},{"key":"ref_22","first-page":"1182","article-title":"Oxidative stress, mitochondrial dysfunction and calcium overload in human lamina cribrosa cells from glaucoma donors","volume":"17","author":"McElnea","year":"2011","journal-title":"Mol. Vis."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1016\/j.exer.2013.07.022","article-title":"Elevated maxi-K+ ion channel current in glaucomatous lamina cribrosa cells","volume":"115","author":"Irnaten","year":"2013","journal-title":"Exp. Eye Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"107980","DOI":"10.1016\/j.exer.2020.107980","article-title":"Transient receptor potential channels TRPC1\/TRPC6 regulate lamina cribrosa cell extracellular matrix gene transcription and proliferation","volume":"193","author":"Irnaten","year":"2020","journal-title":"Exp. Eye Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1146\/annurev.bioeng.9.060906.151927","article-title":"Cell Mechanics: Integrating Cell Responses to Mechanical Stimuli","volume":"9","author":"Janmey","year":"2007","journal-title":"Annu. Rev. Biomed. Eng."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1038\/35036035","article-title":"The versality and universality of calcium signaling","volume":"1","author":"Berridge","year":"2000","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/S0065-230X(08)60765-4","article-title":"Signal Transduction through MAP Kinase Cascades","volume":"74","author":"Lewis","year":"1998","journal-title":"Adv. Cancer Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1351","DOI":"10.1093\/ndt\/gfr550","article-title":"Inhibition of the p38 MAPK pathway ameliorates renal fibrosis in an NPHP2 mouse model","volume":"27","author":"Sugiyama","year":"2011","journal-title":"Nephrol. Dial. Transplant."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1042\/BJ20100323","article-title":"Mechanisms and functions of p38 MAPK signalling","volume":"429","author":"Cuadrado","year":"2010","journal-title":"Biochem. J."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"352371","DOI":"10.1155\/2014\/352371","article-title":"Functional Roles of p38 Mitogen-Activated Protein Kinase in Macrophage-Mediated Inflammatory Responses","volume":"2014","author":"Yang","year":"2014","journal-title":"Mediat. Inflamm."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1248","DOI":"10.1002\/jcp.22448","article-title":"Role of Smad2\/3 and p38 MAP kinase in TGF-\u03b21-induced epithelial-mesenchymal transition of pulmonary epithelial cells","volume":"226","author":"Kolosova","year":"2010","journal-title":"J. Cell. Physiol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"10","DOI":"10.3747\/pdi.2010.00200","article-title":"Activation of p38 Mitogen-Activated Protein Kinase Promotes Peritoneal Fibrosis by Regulating Fibrocytes","volume":"32","author":"Kokubo","year":"2012","journal-title":"Perit. Dial. Int."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1161\/CIRCRESAHA.112.272963","article-title":"Unrestrained p38 MAPK Activation in Dusp1\/4 Double-Null Mice Induces Cardiomyopathy","volume":"112","author":"Accornero","year":"2013","journal-title":"Circ. Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1007\/s11912-012-0227-y","article-title":"PI3K and mTOR Signaling Pathways in Cancer: New Data on Targeted Therapies","volume":"14","author":"Willems","year":"2012","journal-title":"Curr. Oncol. Rep."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2-\u0394\u0394CT Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"4912","DOI":"10.1167\/iovs.08-2192","article-title":"Mitochondrial Defects and Dysfunction in Calcium Regulation in Glaucomatous Trabecular Meshwork Cells","volume":"49","author":"He","year":"2008","journal-title":"Investig. Opthalmol. Vis. Sci."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1089","DOI":"10.1016\/0006-2952(96)00046-9","article-title":"Tissue-specific developmental regulation of protein kinase C isoforms","volume":"51","author":"Goldberg","year":"1996","journal-title":"Biochem. Pharmacol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1016\/j.yjmcc.2010.10.004","article-title":"Protein kinase C\u03b1 as a heart failure therapeutic target","volume":"51","author":"Liu","year":"2011","journal-title":"J. Mol. Cell. Cardiol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1023\/A:1021106232511","article-title":"Alterations in protein kinase C isoenzyme expression and autophosphorylation during the progression of pressure overload-induced left ventricular hypertrophy","volume":"242","author":"Bayer","year":"2003","journal-title":"Mol. Cell. Biochem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"384","DOI":"10.1161\/01.CIR.99.3.384","article-title":"Increased Protein Kinase C Activity and Expression of Ca2+-Sensitive Isoforms in the Failing Human Heart","volume":"99","author":"Bowling","year":"1999","journal-title":"Circulation"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1161\/CIRCRESAHA.107.148288","article-title":"Augmented Protein Kinase C-\u03b1\u2013Induced Myofilament Protein Phosphorylation Contributes to Myofilament Dysfunction in Experimental Congestive Heart Failure","volume":"101","author":"Belin","year":"2007","journal-title":"Circ. Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1038\/nm1000","article-title":"PKC-\u03b1 regulates cardiac contractility and propensity toward heart failure","volume":"10","author":"Braz","year":"2004","journal-title":"Nat. Med."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1303","DOI":"10.1016\/j.neurobiolaging.2014.11.016","article-title":"BDNF prevents amyloid-dependent impairment of LTP in the entorhinal cortex by attenuating p38 MAPK phosphorylation","volume":"36","author":"Criscuolo","year":"2015","journal-title":"Neurobiol. Aging"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"244","DOI":"10.1016\/j.semnephrol.2012.04.003","article-title":"TGF-\u03b2 Signaling via TAK1 Pathway: Role in Kidney Fibrosis","volume":"32","author":"Choi","year":"2012","journal-title":"Semin. Nephrol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"370","DOI":"10.1097\/01.ASN.0000109669.23650.56","article-title":"The Role of p38 Mitogen-Activated Protein Kinase Activation in Renal Fibrosis","volume":"15","author":"Stambe","year":"2004","journal-title":"J. Am. Soc. Nephrol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"11057","DOI":"10.1074\/jbc.272.17.11057","article-title":"Reconstitution of Mitogen-activated Protein Kinase Phosphorylation Cascades in Bacteria","volume":"272","author":"Khokhlatchev","year":"1997","journal-title":"J. Biol. Chem."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1041","DOI":"10.1016\/0092-8674(92)90076-O","article-title":"ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK","volume":"68","author":"Wood","year":"1992","journal-title":"Cell"},{"key":"ref_48","first-page":"6683","article-title":"c-Jun N-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinases","volume":"14","author":"Minden","year":"1994","journal-title":"Mol. Cell. Biol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1016\/0092-8674(92)90361-F","article-title":"Activation of the MAP kinase pathway by the protein kinase raf","volume":"71","author":"Howe","year":"1992","journal-title":"Cell"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"7420","DOI":"10.1074\/jbc.270.13.7420","article-title":"Pro-inflammatory Cytokines and Environmental Stress Cause p38 Mitogen-activated Protein Kinase Activation by Dual Phosphorylation on Tyrosine and Threonine","volume":"270","author":"Raingeaud","year":"1995","journal-title":"J. Biol. Chem."},{"key":"ref_51","first-page":"156","article-title":"The stress-activated protein kinase subfamily of c-Jun kinases","volume":"369","author":"Kyriakis","year":"1994","journal-title":"Nat. Cell Biol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"5097","DOI":"10.1016\/S0021-9258(18)53507-9","article-title":"Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro","volume":"268","author":"Robbins","year":"1993","journal-title":"J. Biol. Chem."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1016\/j.semcancer.2019.07.009","article-title":"PI3K\/Akt\/mTOR inhibitors in cancer: At the bench and bedside","volume":"59","author":"Alzahrani","year":"2019","journal-title":"Semin. Cancer Biol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1038\/nrneph.2016.108","article-title":"Roles of mTOR complexes in the kidney: Implications for renal disease and transplantation","volume":"12","author":"Fantus","year":"2016","journal-title":"Nat. Rev. Nephrol."}],"container-title":["Journal of Clinical Medicine"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2077-0383\/10\/1\/62\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T10:46:34Z","timestamp":1760179594000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2077-0383\/10\/1\/62"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,12,26]]},"references-count":54,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2021,1]]}},"alternative-id":["jcm10010062"],"URL":"https:\/\/doi.org\/10.3390\/jcm10010062","relation":{},"ISSN":["2077-0383"],"issn-type":[{"value":"2077-0383","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,12,26]]}}}