Abstract
We have previously demonstrated a high level of stratifin, also known as 14-3-3 sigma (σ) in differentiated keratinocyte cell lysate and conditioned medium (CM). In this study, we asked the question of whether other 14-3-3 isoforms are expressed in human dermal fibroblasts, keratinocytes, intact dermal and epidermal layers of skin. In order to address this question, total proteins extracted from cultured cells or skin layers were subjected to western blot analysis using seven different primary antibodies specific to well-known mammalian isoforms, β, γ, ε, η, σ, τ, and ζ of 14-3-3 protein family. The autoradiograms corresponding to each isoform were then quantified and compared. The results revealed the presence of very high levels of all seven isoforms in cultured keratinocyte and conditioned medium. With the exception of τ isoform, other 14-3-3 isoforms were also present in intact epidermal layer of normal skin. The profile of 14-3-3 proteins in whole skin was similar to that of epidermis. In contrast, only gamma (γ) 14-3-3 isoform, was present in dermal layer obtained from the same skin sample. On the other hand, cultured fibroblasts express a high level of β, ε, γ and η and a low level of ζ and τ, but not σ isoform. However, the levels of 14-3-3 ε, γ and η were barely detectable in fibroblast conditioned medium. Further, we also used immunohistochemical staining to identify the 14-3-3 isoform expressing cells in human skin sections. The finding revealed different expression profile for each of these isoforms mainly in differentiated keratinocytes located within the layer of lucidum. However, fibroblasts located within the dermal layer did not show any detectable levels of these proteins. In conclusion, all members of 14-3-3 proteins are expressed by cells of epidermal but not dermal layer of skins and that these proteins are mainly expressed by differentiated keratinocytes.
Similar content being viewed by others
References
Johnson-Wint B (1988) Do keratinocytes regulate fibroblast collagenase activities during morphogenesis? Ann NY Acad Sci 548:167–173
Machesney M, Tidman N, Waseem A, Kirby L, Leigh I (1998) Activated keratinocytes in the epidermis of hypertrophic scars. Am J Pathol 152:1133–1141
Nagase H, Woessner JF Jr (1999) Matrix metalloproteinases. J Biol Chem 274:21491–21494
Murphy G, Knauper V, Atkinson S, Butler G, English W, Hutton M, Stracke J, Clark I (2002) Matrix metalloproteinase in arthritic disease. Arthritis Res 4(suppl 3):S39–S49
Ghahary A, Shen YJ, Nedelec B, Wang R, Scott PG, Tredget EE (1996) Collagenase production is lower in post-burn hypertrophic scar fibroblasts than normal fibroblasts and is down-regulated by insulin-like growth factor-1. J Invest Dermatol 106:476–481
Birkedal-Hansen H, Moore WG, Bodden MK, Windsor LJ, Birkedal-Hansen B, DeCarlo A, Engler JA (1993) Matrix metalloproteinases: a review. Crit Rev Oral Biol Med 4(2):197–250
Ghahary A, Karimi-Busheri F, Marcoux Y, Li y, Tredget EE, Taghi kilani R, Li L, Zheng J, Karami A, Keller B, Weinfeld M (2004) Keratinocyte—releasable stratifin functions as a potent collagenase—stimulating factor in fibroblasts. J Invest Dermatol 122:1188–1197
Martens GJ, Piosik PA, Danen EH (1992) Evolutionary conservation of the 14-3-3 protein. Biochem Biophys Res Commun 184:1456–1459
Karimi-Busheri F, Marcoux Y, Tredget EE, Li L, Zhing J, Ghorashi M, Weinfeld M, Ghahary A (2002) Expression of a releasable form of annexin II by human keratinocytes. J Cell Biochem 86:737–747
Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–343
Martin H, Patel Y, Jones D, Howell S, Robinson K, Aitken A (1993) Antibodies against the major isoforms of 14- 3-3 protein. An antibody specific for the N-acetylated amino terminus of a protein. FEBS Lett 331:296–303
Moore BW, Perez VJ (1967) Specific acidic proteins of the nervous system. In: Carlson FD (ed) Physiological and biochemical aspects of nervous integration. Prentice Hall, Englewood Cliffs, NJ, pp 343–359
Ichimura T, Isobe T, Okuyama T, Yamauchi T, Fujisawa H (1987) Brain 14-3-3 protein is an activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca2+calmodulin-dependent protein kinase II. FEBS Lett 219:79–82
Ichimura T, Isobe T, Okuyama T, Takahashi N, Araki K, Kuwano R, Akahashi Y (1988) Molecular cloning of cDNA coding for brain-specific 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases. Proc Natl Acad Sci 85:7084–7088
Toker A, Ellis CA, Sellers LA, Atiken A (1990) Protein kinase C inhibitor proteins. Purification from sheep brain and sequence similarity to lipocortins and 14-3-3 protein. Eur J Biochem 191:421–429
Pozuelo Rubio M, Geraghty KM, Wong BH, Wood NT, Campbell DG, Morrice N, MacKintosh C (2004) 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation, and trafficking. Biochem J 379:395–408
Hermeking H, Lengauer C, Polyak K, He TC, Zhang L, Thiagalingam S, Kinzler KW, Volgelstein B (1997) 14-3-3 sigma is a p53-regulated inhibitor of G2/M progression. Mol Cell 1:3–11
Craparo A, Freund R, Gustafson T (1997) 14-3-3 (epsilon) interacts with the insulin-like growth factor I receptor and insulin receptor substrate I in a phosphoserine-dependent manner. J Biol Chem 272:11663–11669
Chan TA, Hwang PM, Hermeking H, Kinzler KW, Vogelstein B (2000) Cooperative effects of genes controlling the G(2)/M checkpoint. Genes Dev 14:1584–1588
Laronga C, Yang HY, Neal C, Lee MH (2000) Association of the cyclin-dependent kinases and 14-3-3 sigma negatively regulates cell cycle progression. J Biol Chem 275:23106–23112
Yaffe MB (2002) How do 14-3-3 proteins work?—Gatekeeper phosphoraylation and the molecular anvil hypothesis. FEBS Let 513(1):53–57
Chan TA, Hermeking H, Lengauer C, Kinzler KW, Volgelstein B (1999) 14-3-3 sigma is required to prevent mitotic catastrophe after DNA damage. Nature 401:616–620
Ghahary A, Marcoux Y, Karimi-Busheri F, Li Y, Tredget EE, Kilani RT, Lam E, Weinfeld M (2005) Differentiated keratinocyte-releasable stratifin (14-3-3 sigma) stimulates MMP-1 expression in dermanl fibroblasts. J Invest Derm 124(1):170–177
Dellambra E, Golisano O, Bondanza S, Siviero E, Lacal P, Molinari M, D’Atri S, De Luca M (2000) Down regulation of 14-3-3 sigma prevents clonal evolution and leads to immortalization of primary human keratinocytes. J Cell Biol 149(5):1117–1130
Choi KC, Lee S, Kwak SY, Kim MS, Choi HK, Kim KH, Chung JH, Park SH (2005) Increased expression of 14-3-3varepsilon protein in intrinsically aged and photo aged human skin in vivo. Mech Ageing Dev 126(6–7):629–636
Brennan M, Bhatti H, Nerusu KC, Bhagavathula N, Kang S, Fisher GJ, Varani J, Voorhees JJ (2003) Matrix metalloproteinase-1 is the major collagenolytic enzyme responsible for collagen damage in UV-irradiated human skin. Photochem Photobiol 78(1):43–48
Kilani RT, Guilbert L, Lin X, Ghahary A (2007) Keratinocyte conditioned medium abrogates the modulatory effects of IGF-1 and TGF-beta 1 on collagenase expression in dermal fibroblasts. Wound Repair Regen 15(2):236–244
Hsich G, Kenney K, Gibbs CJ, Lee KH, Harrington MG (1996) The 14-3-3 brain protein in cerebrospinal fluid as a marker for transmissible spongiform encephalopathies. N Engl J Med 335(13):924–930
Boston PF, Jackson P, Thompson RJ (1982) Human 14-3-3 proteins: radioimmunoassay, tissue distribution and cerebrospinal fluid levels in patients with neurological disorders. J Neurochem 38:1475–1482
Wiltfang J, Otto M, Baxter HC, Bodemer M, Steinacker P, Bahn E, Kornhuber J, Kretzschmar HA, Poser S, Aitken A (1999) Isoform pattern of 14-3-3 proteins in the cerebrospinal fluid of patients with CJD. J Neurochem 73:2485–2490
Baxter HC, Liu WG, Forster JL, Aitken A, Fraser JR (2002) Immunolocalisation of 14-3-3 isoforms in normal and scrapie-infected murine brain. Neuroscience 109(1):5–14
Hermeking H (2005) Extracellular 14-3-3sigma protein: a potential mediator of epithelial-mesenchymal interactions. J Invest Dermatol 124(1):170–177
Andree HA, Stuart MC, Hermens WT, Reutelingsperger CP, Hemker HC, Frederik PM, Willems GM (1992) Clustering of lipid-bound annexin V may explain its anticoagulant effect. J Biol Chem 267(25):17907–17912
Corradi A, Franzi AT, Rubartelli A (1995) Synthesis and secretion of interleukin-1 alpha and interleukin-1 receptor antagonist during differentiation of cultured keratinocytes. Exp Cell Res 217:355–362
Albuquerque ML, Akiyama SK, Schnaper HW (1998) Basic fibroblast growth factor release by human coronary artery endothelial cells is enhanced by matrix proteins, 17beta-estradiol, and a PKC signaling pathway. Exp Cell Res 245:163–169
Jaye M, Howk R, Burgess W, Ricca GA, Chiu IM, Ravera MW, O’Brien SJ, Modi WS, Maciag T, Drohan WN (1986) Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization. Science 233:541–545
Théry C, Boussac M, Véron P, Ricciardi-Castagnoli P, Raposo G, Garin J, Amigorena S (2001) Proteonomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol 166:7309–7318
Acknowledgment
This research was supported by the grant MOP-13387 from Canadian Institute for Health Research.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kilani, R.T., Medina, A., Aitken, A. et al. Identification of different isoforms of 14-3-3 protein family in human dermal and epidermal layers. Mol Cell Biochem 314, 161–169 (2008). https://doi.org/10.1007/s11010-008-9777-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11010-008-9777-6