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Acetylcholine enhances keratocyte proliferation through muscarinic receptor activation.
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
2015 (English)In: International Immunopharmacology, ISSN 1567-5769, E-ISSN 1878-1705, Vol. 29, no 1, 57-62 p.Article in journal (Refereed) Published
Abstract [en]

Acetylcholine (ACh), a classical neurotransmitter, has been shown to be present in various non-neuronal cells, including cells of the eye, such as corneal epithelium and endothelium, and to have widespread physiological effects such as cytoskeleton reorganization, cellular proliferation, differentiation, and apoptosis. The aim of this study was to investigate the effect of ACh on corneal keratocyte proliferation, and the underlying mechanisms, in order to explore its possible effect in corneal wound healing. Primary culture of human keratocytes was established from donated corneas. Cell viability and fraction of proliferating cells were detected by MTS assay and BrdU incorporation ELISA, respectively. Expression of proliferative markers, PCNA and Ki-67, was detected by western blot and immunocytochemistry. Activation of the MAPK/Erk signaling pathway and its involvement in ACh-enhanced proliferation was determined by western blot analysis, MTS, and BrdU ELISA. We found that ACh enhanced keratocyte proliferation even at low concentrations. Stimulation of proliferation was mediated through activation of muscarinic ACh receptors (mAChRs). Western blot analysis revealed that ACh stimulation of keratocytes upregulated the expression of PCNA and Ki-67, and Ki-67 immunocytochemistry showed that ACh-treated cells were in an active phase of the cell cycle. ACh activated MAPK signaling, and this step was crucial for the ACh-enhanced proliferation, as inhibition of the MAPK pathway resulted in ACh having no proliferative effect. In conclusion, ACh enhances keratocyte proliferation and might thus play a role in proper corneal wound healing.

Place, publisher, year, edition, pages
2015. Vol. 29, no 1, 57-62 p.
Keyword [en]
substance P, acetylcholine, migration, proliferation, apoptosis, corneal stroma
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:umu:diva-112026DOI: 10.1016/j.intimp.2015.05.039ISI: 000365365500010PubMedID: 26049030Scopus ID: 2-s2.0-84946489978OAI: oai:DiVA.org:umu-112026DiVA: diva2:874891
Available from: 2015-11-30 Created: 2015-11-30 Last updated: 2017-12-01Bibliographically approved
In thesis
1. Neuropeptides and neurotransmitters in keratocytes: importance in corneal wound healing processes
Open this publication in new window or tab >>Neuropeptides and neurotransmitters in keratocytes: importance in corneal wound healing processes
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: The cornea is the outermost transparent layer of the eye and it is responsible for the majorityof the eye’s total focusing power. Keratocytes are the resident cells of the corneal stroma and their function isto produce extracellular matrix components and to take part in corneal healing after injury, which may occurdue to trauma, infection or surgery. The process of corneal wound healing is complex. Shortly, keratocytesadjacent to the corneal wound undergo apoptosis and remaining cells start the process of proliferation andmigration in order to close the wound. Next, an influx of inflammatory cells such as macrophages andneutrophils occurs in order to clear the cornea from cellular debris. The final stage of the healing processrestores the quiescent state of keratocytes and remodels any disordered extracellular matrix components,leading to a healthy, transparent cornea. However, when the process of corneal wound healing is incompleteor disturbed, corneal scarring may occur, which can lead to significantly impaired vision. Despite extensiveresearch on corneal wound healing, corneal scarring remains a major cause of preventable blindness. Thehealing process is dependent on various cytokines and growth factors. However, it is possible that also othersignal substances are involved. Substance P (SP) is a neuropeptide well known for its role in pain perception.It has been shown that SP can also be produced by non-neuronal cells, including cells of the cornea, and thatit can have vast effects on physiological functions, including immune cell activity, and cellular processes, suchas cell migration, proliferation, and production of proinflammatory cytokines. Similarly, acetylcholine (ACh),a classical neurotransmitter, has also been reported to be produced by non-neuronal cells, including cornealepithelium, and to be involved in cell proliferation, angiogenesis, cell migration, apoptosis, and collagen geneexpression. In the studies of this thesis, it is hypothesized that neuropeptides and neurotransmitters areproduced by human keratocytes and that this production is increased in response to corneal injury. Moreover,it is hypothesized that the non-neuronal SP and ACh produced by injured keratocytes participate in cornealwound healing by enhancing keratocyte migration and proliferation, and/or by decreasing keratocyteapoptosis. The aims of this thesis project were to test these hypotheses and to study the underlying inter- andintracellular mechanisms of the effects of SP and ACh on keratocytes.Results: Cultured primary cells of the human corneal stroma expressed keratocyte markers (keratocan,lumican, CD34, and ALDH), the tachykinins SP and NKA, catecholamines (adrenaline, noradrenaline anddopamine), ACh, and glutamate. Moreover, the cells expressed neurokinin-1 and -2 receptors (NK-1R andNK-2R), dopamine receptor D2, muscarinic ACh receptors (mAChRs) M1, M3, M4 and M5, and NDMAR1glutamate receptor. Significant differences were observed between expression profiles in cultured keratocytesobtained from central and peripheral cornea. Such differences could also be seen between keratocytescultured under various serum concentrations. Expression and secretion of SP in cultured keratocytes wasincreased in response to injury in vitro. SP enhanced migration of cultured keratocytes through stimulation ofits preferred receptor, the NK-1R, and activation of the phosphatidylinositide 3-kinase and Rac1/RhoApathway and subsequent actin cytoskeleton reorganization and formation of focal adhesion points. Moreover,SP stimulation led to upregulated expression of the proinflammatory and chemotactic cytokine interleukin-8(IL-8), which also contributed significantly to SP-enhanced keratocyte migration and to attractingneutrophils. ACh enhanced keratocyte proliferation in vitro at low concentrations and this stimulation wasmediated through activation of mAChRs and activation of MAPK signalling. Moreover, ACh stimulation led toupregulation of two proliferation markers: PCNA and Ki-67. ACh was also able to protect cultured keratocytesfrom Fas-induced apoptosis, even at low concentrations. Activation of mAChRs was necessary for this latterprocess to occur. ACh reduced caspases 3/7 activation in Fas-treated keratocytes. Inhibition of the PKB/Aktpathway revealed that its activation is essential for mediating the anti-apoptotic effect of ACh in keratocytes.Conclusions: This thesis shows that human keratocytes express an array of neuropeptides (SP, NKA) andneurotransmitters (ACh, adrenaline, noradrenaline, dopamine and glutamate), and their receptors, and thatstimulation of NK-1R by SP and stimulation of mAChRs by ACh lead to keratocyte cellular processes that areknown to be involved in corneal wound healing. Specifically, SP enhances keratocyte migration throughupregulation of IL-8, ACh enhances keratocyte proliferation through activation of the MAPK signallingpathway, and ACh is able to protect keratocytes from apoptosis by activation of the PKB/Akt pathway. Takentogether, these findings suggest that both SP and ACh, if entered at the proper stage, could be beneficial forcorneal wound healing.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2015. 56 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1767
Keyword
substance P, acetylcholine, migration, proliferation, apoptosis, corneal stroma
National Category
Cell Biology
Identifiers
urn:nbn:se:umu:diva-111963 (URN)978-91-7601-385-4 (ISBN)
Public defence
2015-12-21, KB3B1, KBC-huset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2015-11-30 Created: 2015-11-27 Last updated: 2015-11-30Bibliographically approved

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