Abstract
In vivo, melanocytes occupy three-dimensional (3D) space. Nevertheless, most experiments involving melanocytes are performed in a two-dimensional microenvironment, resulting in difficulty obtaining accurate results. Therefore, it is necessary to construct an artificial in vivo–like 3D microenvironment. Here, as a step towards engineering a precisely defined acellular 3D microenvironment supporting the maintenance of human epidermal melanocytes (HEMs), we examined the types of integrin heterodimers that are expressed transcriptionally, translationally, and functionally in HEMs. Real-time PCR and fluorescent immunoassay analyses were used to elucidate the expression of integrin α and β subunit genes at the transcriptional and translational levels, respectively. The functionality of the presumed integrin heterodimers was confirmed using attachment and antibody-inhibition assays. Among the genes encoding 12 integrin subunits (α1, α2, α3, α4, α5, α6, α7, αV, β1, β3, β5, and β8) showing significantly higher transcription levels, proteins translated from the integrin α2, α4, α5, β1, β3, and β5 subunit genes were detected on the surface of HEMs. These HEMs showed significantly increased adhesion to collagen I, fibronectin, laminin, and vitronectin, and functional blockade of the integrin α2 subunits significantly inhibited adhesion to collagen I, fibronectin, and laminin. In addition, there was no significant inhibition of the adhesion to fibronectin or vitronectin in HEMs with functional blockade of the integrin α4, α5, or αV subunits. These results indicate that the active integrin α2β1 heterodimer and the inactive integrin α4, α5, αV, β3, and β5 subunits are all localized on the surface of HEMs.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abshire MY, Thomas KS, Owen KA, Bouton AH (2011) Macrophage motility requires distinct α5β1/FAK and α4β1/paxillin signaling events. J Leukoc Biol 89(2):251–257. https://doi.org/10.1189/jlb.0710395
Bodary SC, McLean JW (1990) The integrin beta 1 subunit associated with the vitronectin receptor alpha v subunit to form a novel vitronectin receptor in a human embryonic kidney cell line. J Biol Chem 265(11):5938–5941
Burleson KM, Casey RC, Skubitz KM, Pambuccian SE, Oegema TR Jr, Skubitz AP (2004) Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers. Gynecol Oncol 93(1):170–181. https://doi.org/10.1016/j.ygyno.2003.12.034
Carsberg CJ, Warenius HM, Friedmann PS (1994) Ultraviolet radiation-induced melanogenesis in human melanocytes. Effects of modulating protein kinase C. J Cell Sci 107(Pt 9):2591–2597
Chiarelli-Neto O, Ferreira AS, Martins WK, Pavani C, Severino D, Faião-Flores F, Maria-Engler SS, Aliprandini E, Martinez GR, di Mascio P, Medeiros MHG, Baptista MS (2014) Melanin photosensitization and the effect of visible light on epithelial cells. PLoS One 9(11):e113266. https://doi.org/10.1371/journal.pone.0113266
Coelho NM, González-García C, Planell JA, Salmerón-Sánchez M, Altankov G (2010) Different assembly of type IV collagen on hydrophilic and hydrophobic substrata alters endothelial cells interaction. Eur Cells Mater 19:262–272
Couchman JR, King JL, McCarthy KJ (1990) Distribution of two basement membrane proteoglycans through hair follicle development and the hair growth cycle in the rat. J Investig Dermatol 94(1):65–70. https://doi.org/10.1111/1523-1747.ep12873363
Danen EH, Ten Berge PJ, Van Muijen GN, Van ‘t Hof-Grootenboer AE, Bröcker EB, Ruiter DJ (1994) Emergence of alpha 5 beta 1 fibronectin- and alpha v beta 3 vitronectin-receptor expression in melanocytic tumour progression. Histopathology 24(3):249–256. https://doi.org/10.1111/j.1365-2559.1994.tb00517.x
Delannet M, Martin F, Bossy B, Cheresh DA, Reichardt LF, Duband JL (1994) Specific roles of the alpha V beta1, alpha V beta 3 and alpha V beta 5 integrins in avian neural crest cell adhesion and migration on vitronectin. Development 120(9):2687–2702
Di Maggio N, Martella E, Frismantiene A, Resink TJ, Schreiner S, Lucarelli E et al (2017) Extracellular matrix and α5β1 integrin signaling control the maintenance of bone formation capacity by human adipose-derived stromal cells. Sci Rep 7:44398. https://doi.org/10.1038/srep44398
Hartman CD, Isenberg BC, Chua SG, Wong JY (2017) Extracellular matrix type modulates cell migration on mechanical gradients. Exp Cell Res 359(2):361–366. https://doi.org/10.1016/j.yexcr.2017.08.018
Ishikawa-Sakurai M, Hayashi M (1993) Two collagen-binding domains of vitronectin. Cell Struct Funct 18(4):253–259
Jokinen J, Dadu E, Nykvist P, Käpylä J, White DJ, Ivaska J, Vehviläinen P, Reunanen H, Larjava H, Häkkinen L, Heino J (2004) Integrin-mediated cell adhesion to type I collagen fibrils. J Biol Chem 279(30):31956–31963. https://doi.org/10.1074/jbc.M401409200
Joshi PG, Nair N, Begum G, Joshi NB, Sinkar VP, Vora S (2007) Melanocyte-keratinocyte interaction induces calcium signalling and melanin transfer to keratinocytes. Pigment Cell Res 20(5):380–384. https://doi.org/10.1111/j.1600-0749.2007.00397.x
Keld O, Donald MS, Jorgen P, Klaus B, Jesper H, Claus BA (1998) Expression of α and β subunits of the integrin superfamily in articular cartilage from macroscopically normal and osteoarthritic human femoral heads. Ann Rheum Dis 57(5):303–308. https://doi.org/10.1136/ard.57.5.303
Kobayashi N, Muramatsu T, Yamashina Y, Shirai T, Ohnishi T, Mori T (1993) Melanin reduces ultraviolet-induced DNA damage formation and killing rate in cultured human melanoma cells. J Investig Dermatol 101(5):685–689. https://doi.org/10.1111/1523-1747.ep12371676
Koohestani F, Braundmeier AG, Mahdian A, Seo J, Bi J, Nowak RA (2013) Extracellular matrix collagen alters cell proliferation and cell cycle progression of human uterine leiomyoma smooth muscle cells. PLoS One 8(9):e75844. https://doi.org/10.1371/journal.pone.0075844
Le Poole IC, Mutis T, van den Wijngaard RM, Westerhof W, Ottenhoff T, de Vries RR, Das PK (1993a) A novel, antigen-presenting function of melanocytes and its possible relationship to hypopigmentary disorders. J Immunol 151(12):7284–7292
Le Poole IC, van den Wijngaard RM, Westerhof W, Verkruisen RP, Dutrieux RP, Dingemans KP, Das PK (1993b) Phagocytosis by normal human melanocytes in vitro. Exp Cell Res 205(2):388–395. https://doi.org/10.1006/excr.1993.1102
Lee ST, Jang M, Lee G, Lim JM (2013) Development of three dimensional culture and expression of integrin heterodimers in human embryonic stem cells. Organogenesis 9(3):143–148. https://doi.org/10.4161/org.25412
Li L, Fukunaga-Kalabis M, Herlyn M (2011) The three-dimensional human skin reconstruct model: a tool to study normal skin and melanoma progression. J Vis Exp 54:2937. https://doi.org/10.3791/2937
Lightner VA, Erickson HP (1990) Binding of hexabrachion (tenascin) to the extracellular matrix and substratum and its effect on cell adhesion. J Cell Sci 95(Pt 2):263–277
Maguer-Satta V, Forissier S, Bartholin L, Martel S, Jeanpierre S, Bachelard E, Rimokh R (2006) A novel role for fibronectin type I domain in the regulation of human hematopoietic cell adhesiveness through binding to follistatin domains of FLRG and follistatin. Exp Cell Res 312(4):434–442. https://doi.org/10.1016/j.yexcr.2005.11.006
Mould AP, Craig SE, Byron SK, Humphries MJ, Jowitt TA (2014) Disruption of integrin-fibronectin complexes by allosteric but not ligand-mimetic inhibitors. Biochem J 464(3):301–313. https://doi.org/10.1042/BJ20141047
Neitmann M, Alexander M, Brinckmann J, Schlenke P, Tronnier M (1999) Attachment and chemotaxis of melanocytes after ultraviolet irradiation in vitro. Br J Dermatol 141(5):794–801. https://doi.org/10.1046/j.1365-2133.1999.03151.x
Neto DS, Pantaleão L, de Sá BC, Landman G (2007) Alpha-v-beta3 integrin expression in melanocytic nevi and cutaneous melanoma. J Cutan Pathol 34(11):851–856. https://doi.org/10.1111/j.1600-0560.2007.00730.x
Okazaki M, Yoshimura K, Uchida G, Harii K (2005) Correlation between age and the secretions of melanocyte-stimulating cytokines in cultured keratinocytes and fibroblasts. Br J Dermatol 153(Suppl 2):23–29. https://doi.org/10.1111/j.1365-2133.2005.06966.x
Popov C, Radic T, Haasters F, Prall WC, Aszodi A, Gullberg D, Schieker M, Docheva D (2011) Integrins α2β1 and α11β1 regulate the survival of mesenchymal stem cells on collagen I. Cell Death Dis 2:e186. https://doi.org/10.1038/cddis.2011.71
Schwartz MA, Assoian RK (2001) Integrins and cell proliferation: regulation of cyclin-dependent kinases via cytoplasmic signaling pathways. J Cell Sci 114(14):2553–2560
Sun T, Jackson S, Haycock JW, MacNeil S (2006) Culture of skin cells in 3D rather than 2D improves their ability to survive exposure to cytotoxic agents. J Biotechnol 122(3):372–381. https://doi.org/10.1016/j.jbiotec.2005.12.021
Sun T, Norton D, McKean RJ, Haycock JW, Ryan AJ, MacNeil S (2007) Development of a 3D cell culture system for investigating cell interactions with electrospun fibers. Biotechnol Bioeng 97(5):1318–1328. https://doi.org/10.1002/bit.21309
Tam I, Stępień K (2011) Secretion of proinflammatory cytokines by normal human melanocytes in response to lipopolysaccharide. Acta Biochim Pol 58(4):507–511
Thingnes J, Lavelle TJ, Hovig E, Omholt SW (2012) Understanding the melanocyte distribution in human epidermis: an agent-based computational model approach. PLoS One 7(7):e40377. https://doi.org/10.1371/journal.pone.0040377
Wayner EA, Orlando RA, Cheresh DA (1991) Integrins alpha v beta 3 and alpha v beta 5 contribute to cell attachment to vitronectin but differentially distribute on the cell surface. J Cell Biol 113(4):919–929. https://doi.org/10.1083/jcb.113.4.919
Yang IJ, Park CH, Kho IS, Lee SJ, Suh KS, Kim TJ (2017) Serosal cavities contain two populations of innate-like integrin α4highCD4+ T cells, integrin α4β1+α6β1+α4β7− and α4β1+α6β1−α4β7+ cells. Immune Netw 17(6):392–401. https://doi.org/10.4110/in.2017.17.6.392
Yebra M, Diaferia GR, Montgomery AM, Kaido T, Brunken WJ, Koch M et al (2011) Endothelium-derived Netrin-4 supports pancreatic epithelial cell adhesion and differentiation through Integrins α2β1 and α3β1. PLoS One 6(7):e22750. https://doi.org/10.1371/journal.pone.0022750
Yu X, Miyamoto S, Mekada E (2000) Integrin α2β1-dependent EGF receptor activation at cell-cell contact sites. J Cell Sci 113(12):2139–2147
Zhou H, Kramer RH (2005) Integrin engagement differentially modulates epithelial cell motility by RhoA/ROCK and PAK1. J Biol Chem 280(11):10624–10635
Funding
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1D1A3B03933811).
Author information
Authors and Affiliations
Contributions
SJK designed and carried out most of the experiments in this research. HJP and HL contributed to the translational analysis of genes and antibody inhibition assay. MSK, JIY, and HWL analyzed and discussed the results. The manuscript was written by SJK, JIY, and STL, and STL supervised the research.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Editor: Tetsuji Okamoto
The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/0blAOM
Electronic supplementary material
ESM 1
(DOCX 2055 kb)
Rights and permissions
About this article
Cite this article
Kim, S.J., Kim, M.S., Park, H.J. et al. Screening of integrins localized on the surface of human epidermal melanocytes. In Vitro Cell.Dev.Biol.-Animal 56, 435–443 (2020). https://doi.org/10.1007/s11626-020-00471-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11626-020-00471-4