C-terminal fragment signaling of membrane-anchored growth factor HB-EGF

肝素结合EGF样生长因子（HB-EGF）最初合成为I型跨膜蛋白（proHB-EGF）。proHB-EGF由特定的金属蛋白酶脱落，将N-末端片段作为可溶性生长因子（HB-EGF）释放到细胞外空间，并将C-末端片段（HB-EGF-C）释放到细胞内空间，在那里它阻止了早幼粒细胞白血病锌指蛋白（PLZF）的转录抑制。本研究的目的是表征proHB-EGF脱落的调节，并研究其在整个细胞周期中HB-EGF-C定位的时间变化。细胞表面proHB-EGF和条件培养基中HB-EGF的定量组合分析表明，proHB-EGF脱落发生在G1细胞周期阶段。激光扫描细胞仪显示HB-EGF-C在G1期晚期被内化到细胞质中，并在S期开始在细胞核中积累。PLZF的后续核输出发生在S期晚期。此外，HB-EGF-C定位在核膜破裂后的中心体周围，并定位在M期晚期染色体分离的带间空间。HB-EGF定位在整个细胞周期的时间变化的特点也是通过延时成像的细胞表达YFP标记的proHB-EGF，这些结果是一致的细胞计数研究中获得的。此外，我们确定BCL-6和BAZF作为HB-EGF-C靶向的转录抑制因子。这些结果表明，proHB-EGF脱落和随后的HB-EGF-C信号与细胞周期的进展，并可能提供一个线索，以了解非受体介导的信号的proHB-EGF在细胞生长中的独特的生物学意义。

Heparin-binding EGF-like growth factor(HB-EGF) is initially synthesized as a type I transmembrane protein (proHB-EGF). The proHB-EGF is shed by specific metalloproteases, releasing the N-terminal fragment into the extracellular space as a soluble growth factor (HB-EGF) and the C-terminal fragment (HB-EGF-C) into the intracellular space, where it prevents transcriptional repression by the promyelocytic leukemia zinc finger protein (PLZF). The goal of the present study was to characterize regulation of proHB-EGF shedding and study its temporal variations in HB-EGF-C localization throughout the cell cycle. Quantitative combination analyses of cell surface proHB-EGF and HB-EGF in conditioned medium showed that proHB-EGF shedding occurred during the G1 cell cycle phase. Laser scanning cytometry revealed that HB-EGF-C was internalized into the cytoplasm during the late G1 phase and accumulated in the nucleus beginning in the S phase. Subsequent nuclear export of PLZF occurred during the late S phase. Further, HB-EGF-C was localized around the centrosome following breakdown of the nuclear envelope and was localized to the interzonal space with chromosome segregation in the late M phase. Temporal variations in HB-EGF localization throughout the cell cycle were also characterized by time-lapse imaging of cells expressing YFP-tagged proHB-EGF, and these results were consistent with those obtained in cytometry studies. Furthermore, we identified BCL-6 and BAZF as transcriptional repressors targeted by HB-EGF-C. These results indicate that proHB-EGF shedding and subsequent HB-EGF-C signaling are related with progression of the cell cycle and may provide a clue to understand the unique biological significance of non-receptor-mediated signaling of proHB-EGF in cell growth.