Paediatric Endocrine Research Laboratory, Division of Paediatric Endocrinology and Diabetes, Department of Paediatrics, University of Patras School of Medicine, Patras 26500, Greece, Phone: +30 6972070117, Fax: +30 2610993462.
Faculty of Human Movement and Quality of Life Sciences Department of Nursing, University of Peloponnese, Sparta, Greece.
Horm Mol Biol Clin Investig. 2020 Mar 2;41(2):hmbci-2019-0029. doi: 10.1515/hmbci-2019-0029.
Background Growth hormone(GH) and epidermal growth factor (EGF) stimulate cell growth and differentiation, and crosstalking between their signaling pathways is important for normal cellular development. Growth hormone transduction defect (GHTD) is characterized by excessive GH receptor (GHR) degradation, due to over-expression of the E3 ubiquitin ligase, cytokine inducible SH2-containing protein (CIS). GH induction of GHTD fibroblasts after silencing of messenger RNA (mRNA) CIS (siCIS) or with higher doses of GH restores normal GH signaling. β-Transducing-repeat-containing protein (β-TrCP), another E3 ubiquitin ligase, also plays a role in GHR endocytosis. We studied the role of β-TrCP in the regulation of the GH/GHR and EGF/EGF receptor (EGFR) pathways in normal and GHTD fibroblasts. Materials and methods Fibroblast cultures were developed from gingival biopsies of a GHTD (P) and a control child (C). Protein expression and cellular localization of β-TrCP were studied by Western immunoblotting and immunofluorescence, respectively, after: (1) GH 200 μg/L human GH (hGH) induction, either with or without silence CIS (siCIS), and (2) inductions with 200 μg/L GH or 1000 μg/L GH or 50 ng/mL EGF. Results After induction with: (1) GH200/siCIS, the protein expression and cytoplasmic-membrane localization of β-TrCP were increased in the patient, (2) GH200 in the control and GH1000 in the patient, the protein and cytoplasmic-membrane localization of β-TrCP were increased and (3) EGF, the protein expression and cytoplasmic-membrane localization of β-TrCP were increased in both the control and the patient. Conclusions (1) β-TrCP appears to be part of the negative regulatory mechanism of the GH/GHR and EGF/EGFR pathways. (2) There appears to be a negative correlation between β-TrCP and CIS. (3) In the control and GHTD patient, β-TrCP increases when CIS is suppressed, possibly as a compensatory inhibitor of the GH/GHR pathway.
生长激素（GH）和表皮生长因子（EGF）刺激细胞生长和分化，其信号通路之间的串扰对于正常细胞发育很重要。生长激素转导缺陷（GHTD）的特征是由于细胞因子诱导的 SH2 结构域蛋白（CIS）的过度表达导致 GH 受体（GHR）的过度降解。沉默信使 RNA（mRNA）CIS（siCIS）或用更高剂量 GH 诱导 GHTD 成纤维细胞后，GH 可恢复正常 GH 信号。另一种 E3 泛素连接酶β-转化重复包含蛋白（β-TrCP）也在 GHR 内吞中起作用。我们研究了β-TrCP 在正常和 GHTD 成纤维细胞中 GH/GHR 和 EGF/EGF 受体（EGFR）途径调节中的作用。
从 GHTD（P）和对照儿童（C）的牙龈活检中开发了成纤维细胞培养物。通过 Western 免疫印迹和免疫荧光分别研究了β-TrCP 的蛋白表达和细胞内定位，方法如下：（1）用或不用沉默 CIS（siCIS）用 200μg/L 人 GH（hGH）诱导 GH，（2）用 200μg/L GH 或 1000μg/L GH 或 50ng/mL EGF 诱导。
用以下方法诱导后：（1）GH200/siCIS，患者中β-TrCP 的蛋白表达和细胞质膜定位增加，（2）GH200 在对照中，GH1000 在患者中，β-TrCP 的蛋白和细胞质膜定位增加，（3）EGF，对照和患者中β-TrCP 的蛋白表达和细胞质膜定位增加。
（1）β-TrCP 似乎是 GH/GHR 和 EGF/EGFR 途径负调节机制的一部分。（2）β-TrCP 与 CIS 之间似乎存在负相关。（3）在对照和 GHTD 患者中，当 CIS 被抑制时，β-TrCP 增加，可能作为 GH/GHR 途径的补偿性抑制剂。
