Dieckow Julia, Brandt Wolfgang, Hattermann Kirsten, Schob Stefan, Schulze Ute, Mentlein Rolf, Ackermann Philipp, Sel Saadettin, Paulsen Friedrich P
Department of Ophthalmology University of Leipzig, Leipzig, Germany 2Department of Anatomy II, Friedrich Alexander University Erlangen-Nürnberg, Erlangen, Germany.
Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle, Germany.
Invest Ophthalmol Vis Sci. 2016 Jan 1;57(1):56-65. doi: 10.1167/iovs.15-18129.
Trefoil factor family (TFF) peptides, and in particular TFF3, are characteristic secretory products of mucous epithelia that promote antiapoptosis, epithelial migration, restitution, and wound healing. For a long time, a receptor for TFF3 had not yet been identified. However, the chemokine receptor CXCR4 has been described as a low affinity receptor for TFF2. Additionally, CXCR7, which is able to heterodimerize with CXCR4, has also been discussed as a potential TFF2 receptor. Since there are distinct structural similarities between the three known TFF peptides, this study evaluated whether CXCR4 and CXCR7 may also act as putative TFF3 receptors.
We evaluated the expression of both CXCR4 and CXCR7 in samples of human ocular surface tissues and cell lines, using RT-PCR, immunohistochemistry, and Western blot analysis. Furthermore, we studied possible binding interactions between TFF3 and the receptor proteins in an x-ray structure-based modeling system. Functional studies of TFF3-CXCR4/CXCR7 interaction were accomplished by cell culture-based migration assays, flow cytometry, and evaluation of activation of the mitogen-activated protein (MAP) kinase signaling cascade.
We detected both receptors at mRNA and protein level in all analyzed ocular surface tissues, and in lesser amount in ocular surface cell lines. X-ray structure-based modeling revealed CXCR4 and CXCR7 dimers as possible binding partners to TFF3. Cell culture-based assays revealed enhanced cell migration under TFF3 stimulation in a conjunctival epithelial cell line, which was completely suppressed by blocking CXCR4 and/or CXCR7. Flow cytometry showed increased proliferation rates after TFF3 treatment, while blocking both receptors had no effect on this increase. Trefoil factor family 3 also activated the MAP kinase signaling cascade independently from receptor activity.
Dimers CXCR4 and CXCR7 are involved in TFF3-dependent activation of cell migration, but not cell proliferation. The ERK1/2 pathway is activated in the process, but not influenced by CXCR4 or CXCR7. These results implicate a dependence of TFF3 activity as to cell migration on the chemokine receptors CXCR4 and CXCR7 at the ocular surface.
三叶因子家族（TFF）肽，尤其是TFF3，是黏液上皮的特征性分泌产物，可促进抗凋亡、上皮迁移、修复和伤口愈合。长期以来，TFF3的受体尚未被鉴定出来。然而，趋化因子受体CXCR4已被描述为TFF2的低亲和力受体。此外，能够与CXCR4异源二聚化的CXCR7也被讨论为潜在的TFF2受体。由于三种已知的TFF肽之间存在明显的结构相似性，本研究评估了CXCR4和CXCR7是否也可能作为假定的TFF3受体。
我们使用逆转录聚合酶链反应（RT-PCR）、免疫组织化学和蛋白质印迹分析，评估了人眼表组织和细胞系样本中CXCR4和CXCR7的表达。此外，我们在基于X射线结构的建模系统中研究了TFF3与受体蛋白之间可能的结合相互作用。通过基于细胞培养的迁移试验、流式细胞术和对丝裂原活化蛋白（MAP）激酶信号级联激活的评估，完成了TFF3-CXCR4/CXCR7相互作用的功能研究。
我们在所有分析的眼表组织中均检测到了两种受体的mRNA和蛋白水平，在眼表细胞系中的表达量较少。基于X射线结构的建模显示，CXCR4和CXCR7二聚体可能是TFF3的结合伙伴。基于细胞培养的试验显示，在结膜上皮细胞系中，TFF3刺激可增强细胞迁移，而阻断CXCR4和/或CXCR7可完全抑制这种迁移。流式细胞术显示，TFF3处理后细胞增殖率增加，而阻断两种受体对这种增加没有影响。三叶因子家族3还独立于受体活性激活了MAP激酶信号级联。
CXCR4和CXCR7二聚体参与了TFF3依赖性的细胞迁移激活，但不参与细胞增殖。在此过程中ERK1/2途径被激活，但不受CXCR4或CXCR7的影响。这些结果表明，眼表TFF3对细胞迁移的活性依赖于趋化因子受体CXCR4和CXCR7。
