Liu J J, Shay J W, Wilson S E
Eye Institute and Department of Cell Biology, The Cleveland Clinic Foundation, Ohio, USA.
Invest Ophthalmol Vis Sci. 1998 Dec;39(13):2584-93.
Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family FGF-7. It exhibits potent mitogenic activity for epithelial cells, including corneal and mammary epithelial cells. A messenger RNA has been reported that is generated by alternative splicing of bek that putatively codes only for the extracellular ligand-binding domain of KGF receptor (soluble KGF receptor). In the present study, the expression of the mRNA coding for this alternative bek transcript was examined and the corresponding protein characterized.
Alternative messenger RNA transcripts were detected in various cell lines or tissues using reverse transcription-polymerase chain reaction (RT-PCR) and RNase protection assay. NIH/3T3 fibroblast cells and 293 kidney embryonic epithelial cells were stably transfected with soluble KGF receptor cDNA and transmembrane KGF receptor cDNA. Soluble KGF receptor protein was produced using a baculovirus-insect expression system. Soluble KGF receptor protein was detected using western and dot blot analyses. Binding assays and cross-linking labeling were used to determine the affinity and specificity of soluble KGF receptor. A mitogenic assay was performed to examine the function of the soluble KGF receptor.
The soluble KGF receptor mRNA was primarily expressed in epithelial cells, including cells from the cornea and breast. Cross-linking labeling and affinity-binding assays with 125I-KGF showed that the soluble KGF receptor bound KGF (FGF-7) but not FGF-1 or FGF-2. Soluble KGF receptor was detected in the culture medium of cells stably transfected with soluble KGF receptor cDNA but not with transmembrane KGF receptor cDNA, suggesting that the soluble receptor was generated by mRNA splicing and probably not by proteolysis or posttranslational processing. Soluble KGF receptor inhibited KGF binding to transmembrane KGF receptor and DNA synthesis in BALB/MK epidermal keratinocytes in response to KGF, suggesting that soluble KGF receptor expression could provide a mechanism for the cell to downregulate responses to KGF.
A truncated soluble KGF receptor expressed in corneal and other epithelial cells probably functions to downregulate the response of the cell to KGF.
角质形成细胞生长因子（KGF）是成纤维细胞生长因子（FGF）家族FGF - 7的成员。它对包括角膜和乳腺上皮细胞在内的上皮细胞具有强大的促有丝分裂活性。据报道，一种信使核糖核酸（mRNA）是由bek基因的可变剪接产生的，推测其仅编码KGF受体的细胞外配体结合域（可溶性KGF受体）。在本研究中，检测了编码这种可变bek转录本的mRNA的表达，并对相应蛋白质进行了表征。
使用逆转录 - 聚合酶链反应（RT - PCR）和核糖核酸酶保护试验在各种细胞系或组织中检测可变信使核糖核酸转录本。用可溶性KGF受体cDNA和跨膜KGF受体cDNA稳定转染NIH/3T3成纤维细胞和293肾胚胎上皮细胞。使用杆状病毒 - 昆虫表达系统产生可溶性KGF受体蛋白。使用蛋白质免疫印迹和斑点印迹分析检测可溶性KGF受体蛋白。结合试验和交联标记用于确定可溶性KGF受体的亲和力和特异性。进行促有丝分裂试验以检查可溶性KGF受体的功能。
可溶性KGF受体mRNA主要在上皮细胞中表达，包括来自角膜和乳腺的细胞。用125I - KGF进行的交联标记和亲和结合试验表明，可溶性KGF受体结合KGF（FGF - 7），但不结合FGF - 1或FGF - 2。在稳定转染可溶性KGF受体cDNA而非跨膜KGF受体cDNA的细胞培养基中检测到可溶性KGF受体，这表明可溶性受体是由mRNA剪接产生的，可能不是由蛋白水解或翻译后加工产生的。可溶性KGF受体抑制KGF与跨膜KGF受体的结合以及BALB/MK表皮角质形成细胞中KGF诱导的DNA合成，这表明可溶性KGF受体的表达可能为细胞下调对KGF的反应提供一种机制。
在角膜和其他上皮细胞中表达的截短型可溶性KGF受体可能起到下调细胞对KGF反应的作用。
