The Expression And Function Of Vascular Endothelial Growth Factor Receptor 2 On HaCaT Cells

Vascular endothelial growth factor (VEGF) is a family of growth factors, including VEGF-A, -B, -C, -D, and -E, and placental growth factor (PlGF). VEGF has been studied extensively for its angiogenic behavior in physiological and pathological conditions.VEGF-A, also named VEGF, is considered as the most important factor among VEGF family. According to the different slicing pattern during VEGF transcription course, there are some different VEGF isotypes including VEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉, VEGF₂₀₆. Keratinocyte is considered as one of the major cell types which could secrete VEGF. In some inflammatory skin diseases, such as psoriasis and atopic dermatitis, there are more VEGF secreted by keratinocytes and play a critical role in angiogenesis in a paracrine pattern. VEGF₁₂₁ and VEGF₁₆₅ are two major isoforms secreted by keratinocytes and VEGF₁₆₅ is the most effective one in them.VEGF family induce the proliferation of endothelial cells, promote angiogenesis and enhance the vascular permeability by binding to VEGF receptors. Vascularendothelial growth factor (VEGF) plays an important role in normal and pathological angiogenesis. VEGF receptors (VEGFRs, including VEGFR-1, VEGFR-2, and VEGFR-3) and neuropilins (NRPs, including NRP-1 and NRP-2) are high-affinity receptors for VEGF and are typically considered to be specific for endothelial cells. VEGFRs belong to the receptor tyrosine kinase (RTK) family and have a characteristic structure with 7 Ig-like domains in the extracellular domain and a cytoplasmic tyrosine kinase domain with a long kinase insert region. VEGF also interacts with neuropilins (NRPs), a family of non-tyrosine kinase transmembrane receptors with a small cytoplasmic domain and multiple extracellular domains. VEGFR-2 is the major receptor for endothelial cell function. Recently, VEGF receptors were found to be expressed not only on the endothelial cells but also on some non-endothelial cells, such as hematopoietic stem cells, neuronal cells, retinal progenitor cells, as well as on some tumor cells. In our lab, we ever detected the expression of VEGFR-1 and VEGFR-2 on normal keratinocytes, hair follicles, and sebaceous glands, and we found that the expression of VEGFR-1 and VEGFR-2 on psoriatic keratinocytes were elevated. Recently, the expression of VEGFR-1 but VEGFR-2 on human and mouse keratinocytes was detected with immunohistology by Wilgus et al. Kurschat et al did not observe the expression of VEGFR-1 and VEGFR-2 in cultured HaCaT cells. Confirmation of the expression of VEGF receptors on keratinocytes will contribute to the investigation of the biology of them in keratinocytes.[Objective]Our aim in this paper is to confirm the expression of VEGF receptors on keratinocytes and investigate whether VEGF secreted by keratinocytes could act in an autocrine pattern by binding to VEGF receptors on themselves, besides the paracrinepattern by binding to VEGF receptors on vascular endothelial cells. Furthermore, we will investigate the role of VEGFR-2 in the autocrine effect of VEGF on keratinocytes. By the way, the effect of tacrolimus on the proliferation of keratinocytes and the expression of VEGF and VEGFR-2 will be investigated. [methods]1. RT-PCR, Western-blot and indirect immunofluorescent staining were used to determine the mRNA and protein expression of the vascular endothelial growth factor receptors, including VEGFR-1, VEGFR-2, VEGFR-3 and neuropilin-1 (NRP-1), neuropilin-2 (NRP-2 ),on HaCaT cell line.2. MTT assays were used to determine the effect of different concentrations of VEGF₁₆₅ and bevacizumab on the proliferation and adhesion abilities of HaCaT cells. Migration assays were used to determine the effect of different concentrations of VEGF₁₆₅ and bevacizumab on the migration of HaCaT cells. Neutralizing antibody to VEGFR-2, MAB3571, was used to determine the role of VEGFR-2 in the effect of VEGF on proliferation, migration and adhesion of HaCaT cells. The effect of VEGF₁₆₅ at 10ng/ml on phosphorylation of VEGFR-2 , PLC-γ1, ERK1/2 were detected in HaCaT cells pretreated with or without MAB3571 at 5μg/ml. The apoptosis of HaCaT cells was determined when the cells were pretreated with 0.5mg/ml bevacizumab or 5μg/ml MAB3571 to inhibite the effect of endogenous VEGF.3. RT-PCR and Western-blot were used to determine the effect of different concentrations of tacrolimus on the mRNA and protein expression of VEGF and VEGFR-2. MTT assays were used to determine the effect of different concentrations of tacrolimus on the proliferation of HaCaT cells, and 10ng/ml VEGF₁₆₅ was used to partly reverse the inhibition effect of tacrolimus on the proliferation of HaCaT cells.[Results]1. The mRNA and protein expression of the vascular endothelial growth factor receptors, including VEGFR-1, VEGFR-2, VEGFR-3 and neuropilin-1 (NRP-1), neuropilin-2 (NRP-2 ), on HaCaT cell line were confirmed by RT-PCR, Western-blot and indirect immunofluorescent staining.2. VEGF₁₆₅ enhanced the proliferation of HaCaT cells in a dose-dependent manner, while bevacizumab could inhibit the proliferation of HaCaT cells in a dose-dependent manner.3. Neutralizing antibody to VEGFR-2, MAB3571, could down-regulate the enhanced proliferation of HaCaT cells by 10ng/ml VEGF₁₆₅.4. VEGF₁₆₅ enhanced the migration of HaCaT cells in a dose-dependent manner, while bevacizumab could inhibit the migration of HaCaT cells in a dose-dependent manner.5. Neutralizing antibody to VEGFR-2, MAB3571, could down-regulate the enhanced migration of HaCaT cells by 10ng/ml VEGF₁₆₅.6. VEGF₁₆₅ inhibited the adhesion ability of HaCaT cells in a dose-dependent manner, while bevacizumab could reverse and promote the adhesion ability of HaCaT cells in a dose-dependent manner.7. Neutralizing antibody to VEGFR-2, MAB3571, could reverse the inhibited adhesion ability of HaCaT cells by 10ng/ml VEGF₁₆₅.8. 10ng/ml VEGF₁₆₅ induced the phosphorylation of VEGFR-2, PLC-γ1, ERK1/2 in HaCaT cells, which were inhibited by pretreated with neutralizing antibody to VEGFR-2, MAB3571, at 5μ/ml9. Inhibition of the endogenous VEGF with 0.5mg/ml bevacizumab or 5μg/ml MAB3571 contribute to the apoptosis of HaCaT cells.10. VEGF₁₆₅ prmote the mRNA and protein level of VEGFR-2 in a dose dependent manner at low concentration.11. Tacrolimus inhibite the mRNA and protein expression of VEGF and VEGFR-2 on HaCaT cells in a dose-dependent manner.12. Tacrolimus inhibite the proliferation of HaCaT cells in a dose dependent manner, which could be partly reversed by 10ng/ml VEGF₁₆₅.[Conclusions]1. The five identified vascular endothelial growth factor receptors, including VEGFR-1, VEGFR-2, VEGFR-3 and neuropilin-1 (NRP-1), neuropilin-2 (NRP-2) are all expressed by ketatinocytes.2. VEGF secreted by keratinocytes acts in an autocrine pattern by binding to VEGF receptors on themselves, besides the paracrine pattern by binding to VEGF receptors on vascular endothelial cells. VEGFR-2 plays a critical role in the autocrine effect of VEGF on keratinocytes.3. The inhibition effect of Tacrolimus on the proliferation of keratinocytes was partly due to the inhibition effect of tacrolimus on the VEGF/VEGFR-2 autocrine effect in keratinocytes.