The Role Of Sprouty1in Human Normal Epidermal Keratinocytes

BACKGROUNDSprouty (SPRY) proteins are a highly conserved group of negative feedback loop modulators of the fibroblast growth factor (FGF)-Ras-ERK signaling pathway activation and were originally discovered in a gene screen in Drosophila responsible for shaping the developing trachea. Mammalian SPRY proteins comprise of four orthologs, SPRY1,2,3and4, whose modulation of RTK-induced signaling pathway is growth factor-and cell context-dependent. Subsequent work established the expression of SPRY1,2, and4being widespread in embryonic and adult tissues, whereas expression of SPRY3being restricted to brain and testes in adult tissues. Receptor tyrosine kinases (RTKs) control a wide variety of cell processes in multicellular organisms, including cell proliferation, differentiation, migration and survival. Cell process is tightly and procisely controlled by the coordinated action of both positive and negative regulators that function at multiple or different levels of the signal transduction cascades, and spatiotemporally within the growth-factor-induced responses. When this process goes awry, the outcome can be developmental defects and malignancy. Sprouty1(SPRY1) represents a major class of ligand-inducible inhibitors of RTK-dependent signaling pathways and is regulated by multiple growth factors. New biochemical and genetic evidences indicate specific and critical roles of the SPRY1gene in development and multiple modes of action of the SPRY1in regulation of the RTK-induced response. Therefore, SPRY1is considered to play a role in the negative feedback loop in multiple cell processes.In several malignancies their expression is deregulated. Loss of SPRY1causes specific phenotypes similar to those observed in case of growth factor overdose. In addition, SPRY1may affect tumor cell function through direct interaction with urokinase plasminogen activator receptor and promote its lysosomal degradation. SPRY1is down-regulated in a variety of cancer types, including breast, prostate, liver, lung and thyroid cancers, especially in metastatic malignant stages involving EMT. Therefore, SPRY1was considered to be a tumor suppressor in the development of malignant tumor.Despite recent advances in understanding the roles of SPRY1on the regulation of FGF receptor signaling during embryogenesis and in the development of malignancy, the role of SPRY1in human skin remains widely unknown.OBJECTIVESince the inhibitory function of SPRY1is cell and ligand specific, the role of SPRY1for differentiation, proliferation and cell progress of keratinocytes in human epidermis is so far incompletely understood. The aim of this study was to investigate the expressive pattern and the effects of SPRY1exerts in epidermal keratinocytes, to investigate whether SPRY1affects the cell progression of human normal epidermal keratinocytes (NHEK). In further, to investigate whether SPRY1would influence the wound healing, imiquimod induced psoriasis-like lesion, contact hypersensitivity and acute inflammatory responses in K14-SPRY1-eGFP transgenic mouse model in vivo.METHODSFirstly, SPRY1expression patterns in healthy human skin and in cultured NHEK were studied by immunofluorescence.Secondly, to determine the role of SPRY1in keratinocytes, forced overexpression SPRY1in vitro was established in NHEK and HaCaT cells by infection with lentivirus, which constructed with human SPRY1gene. The condition of overexpression of SPRY1 in NHEK and HaCaT cells was confirmed by RT-PCR and western blot. Subsequently, by those MAPK, PI3K-AKT, TGF-β/smad signaling pathways and pertinent downstream molecules involving differentiation, proliferation, apoptosis and epithelial mesenchymal transtion of keratinocytes were detected by Western blot, such as p21, p27, cyclin B1and loricrin. HaCaT cells with overexpression SPRY1were utilized to determine the alteration of proliferation, apoptosis and invasion capability.Thirdly, epidermal limited co-localized overexpressed SPRY1transgenic mice were established (K14-SPRY1-eGFP) and generated a series mimic models, including5%imiquimod cream induced psoriasis-like lesion, wound healing,3%oxazolone solution induced contact hypersensitivity and irritants, TPA and CA were induced acute inflammation model, to investigate the functions of SPRY1in vivo. H&E and Masson’s tri-chrome staining were utilized to exhibit and compare the condition of inflammation in the pinnas.RESULTSⅠ. The expression and distribution of SPRY1in normal human epidermis and NHEK1. The expression of SPRY1was limited in the granular layer of the normal human epidermis.2. In cultured NHEK, expression of SPRY1was sparsely and in a very lower level cytoplasmically.Ⅱ. The effects of SPRY1in NHEK and HaCaT cell in vitro1. The forced overexpression of SPRY1was successfully established in NHEK and HaCaT cells by infection with lentivirus, which were constructed with human SPRY1gene;2. Overexpression of SPRY1resulted in up-regulation of P21, P27and down-regulation of cyclin B1and G0S2, these results indicated these alteration would contribute to the alteration of cell cycle and proliferation in NHEK and HaCaT cells;3. SPRY1altered skin barrier and later differentiation, but not changed the earlier differentiation of NHEK. SPRY1up-regulated AQP7and laminin5, but filaggrin, keratin10(K10) and K1, involucrin were not changed, while loricrin was increased by overexpression of SPRY1;4. Regulation of epithelial and mesenchymal markers by SPRY1in NHEK. The levels of vimentin, E-cadherin, N-cadherin and β-catenin were not changed. However, PAI-lwas up-regulated, integrin α6and MMP3were decreased, under the SPRY1overexpressed in NHEK;5. The poly comb group (PcG) proteins were modified by SPRY1. Ring1B was slightly ameliorated by SPRY1. However, EZH2and Bmil were decreased obviously, SUZ12was increased upon overexpression of SPRY1in the NHEK;6. Histone methylation and acetylation upon overexpression of SPRY1in NHEK. The expression of H3K4me2(lysine4,lysine27,lysine9), were evidently increased. However, H3K79me2(lysine79), H3K36me2(lysine36) and H3me3(histone H3tri-methylation) were not altered. The levels of total H2A, H2B, H3and H4, and the acetylated H2A (AcH2A), H2B (AcH2B), H3(AcH3) and H4(AcH4) were all not altered notably. HDAC1,2,3,4and6were also not obviously altered. However, the phosphorylated HDAC4/5/7was decreased remarkably upon SPRY1overexpression in NHEK;7. The levels of HMG family proteins were all not altered notably under the forced overexpression of SPRY1in NHEK;8. The level of phospho-ERK/ERK, phospho-AKT/AKT, phospho-PTEN/PTEN and phospho-Smadl,3/Smad1,3,6,7were not fluctuated in SPRY1overexpressed NHEK;9. Ectopic expression of SPRY1inhibited cell proliferation, migration and senescence, but promoted cell apoptosis of HaCaT cells.Ⅲ. Establishment of K14-SPRYl-eGFP transgenic mice and effects of SPRY1in vivo1. SPRY1transgenic mice exhibited alleviated IMQ-induced psoriatic lesion and delayed skin wound healing;2. Overexpression of SPRY1eased the contact hypersensitivity in vivo;3. Forced overexpression of SPRY1exhibited no prominent differences compared with wild type mice on the acute inflammation in vivo. CONCLUSIONSⅠ. SPRY1was limited localized in the granular layer of the normal human epidermis.Ⅱ. In cultured NHEK, expression of SPRY1was sparsely and in a very lower level cytoplasmically.Ⅲ. SPRY1up-regulated P21, P27and down-regulated cyclin B1and G0S2, these results indicated these alteration would contribute to the alteration of cell cycle and proliferation;Ⅳ. SPRY1up-regulated AQP7, laminin5and loricrin, those changes might contribute to the alteration of skin barrier and later differentiation.Ⅴ. SPRY1up-regulated PAI-1, decreased integrin a6and MMP3, which might limit the cell migration and epithelial mesenchymal transmition in NHEK.Ⅵ.The poly comb group (PcG) proteins, including Ring1B, EZH2and Bmilwere decreased, SUZ12were increased upon overexpression of SPRY1in the NHEK;Ⅶ. The expression of H3K4me2(lysine4,lysine27,lysine9), were increased and the level of phosphorylated HDAC4/5/7was decreased upon SPRY1overexpression in NHEK;Ⅷ.The level of HMG family proteins were all not altered notably under the forced overexpression of SPRY1in NHEK;Ⅸ. Ectopic expression of SPRY1inhibited cell proliferation, migration and senescence, but promoted cell apoptosis of HaCaT cells.Ⅹ. SPRY1transgenic mice exhibited alleviated IMQ-induced psoriatic lesion and delayed skin wound healing;Ⅺ. Overexpression of SPRY1eased the contact hypersensitivity in vivo;Ⅻ. No prominent differences exhibited between SRPY1TG and wild type mice on the acute inflammation in vivo.