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Title: CD105 expression and its effects on TGF-beta signalling, angiogenesis and cell behaviour
Author: Guo, Baoqiang
ISNI:       0000 0004 2674 9853
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2003
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CD 105 (endoglin), a 180kDa homodimeric transmembrane glycoprotein, is highly expressed in angiogenic endothelial cells (ECs) and is an important component of the transforming growth factor-beta (TGF-beta) receptor complex. In an attempt to understand the regulation of CD 105 expression and the molecular mechanism by which it exerts its effect on angiogenesis, human dermal microvascular endothelial cells (HDMECs) and CD 105 transfected rat myoblasts were utilised as an in vitro model with the following results: (1) TGF-beta1 increased CD105 expression in HDMECs at the transcriptional level. Flow cytometry. Western and Northern blot analysis showed that CD 105 expression was increased in HDMECs. When pXP2 plasmid DNA containing a full-length CD 105 promoter was transiently transfected into HDMECs, a marked response to TGF-beta1 was observed, implying that TGF-beta1 upregulates CD 105 expression at the transcriptional level. In contrast, TNF-alpha reduced CD 105 protein in HDMECs independent of its transcriptional regulation. (2) CD105 expression was increased by CoCl2 and overexpression of CD105 regulated HIF-1 transcriptional activity. CD 105 expression in HDMECs was demonstrated by flow cytometry and Western blot analysis to be increased by C0CI2 which is a specific inducer of hypoxia inducible factor-1 (HIF-1). pXP2 plasmids containing CD 105 promoters differing in length, were transiently transfected into HDMECs. They all showed similar luciferase activity in response to CoCl2, implying that the HIF-1 binding site is located in the shortest promoter sequence (-50/+350). Only one 5'-ACGTG-3' consensus sequence was observed in it, and this was responsible for HIF-1 binding and transcriptional activation in response to CoCl2. A hypoxia response element (HRE)-containing plasmid, (HRE)3-Luc, was transiently transfected into mock and CD 105 rat myoblast transfectants. It was demonstrated that overexpression of CD 105 increased luciferase activity in response to CoCl2, suggesting that overexpression of CD 105 increased HIF-1 transcriptional activity. (3) CD105 was involved in proliferation of ECs. A reduction in CD105 expression by CD 105 antisense oligodeoxynucleotide (AS-ODN) led to inhibition of EC proliferation, suggesting that CD 105 expression in HDMECs was involved in the regulation of their proliferation. (4) CD105 modulated TGF-beta1 signalling through both Smad3 and JNK signalling pathways. Overexpression of CD 105 in rat myoblast transfectants antagonised TGF-beta1 mediated inhibition of cell proliferation and reduced TGF-beta1-mediated p3TP-Lux (PAI-1 promoter) luciferase and (CAGA)12-Luc luciferase activity. The CAGA sequence is specific for SmadS and Smad4 binding, implying that CD 105 is involved in inhibition of TGF-beta1/ SmadS signalling. As demonstrated in immunoprecipitation and translocation assays, CD 105 overexpression reduced phosphorylation and nuclear translocation of SmadS. CD 105 expression also resulted in high phosphorylation of JNK1, which is able to activate c-Jun. It is known that c-Jun inhibits transcriptional activity of SmadS on CAGA sites, implying that CD 105 may also inhibit the transcriptional activity of SmadS through JNK. (5) Overexpression of CD105 in rat myoblasts was involved in cell morphology, spreading and persistent directional movement. Mock and CD 105 rat myoblast transfectants were cultured in un-coated plastic dishes in DMEM medium supplemented with 10% FCS. Mock cells displayed polygonal morphology. CD 105 transfectants showed fibroblastoid elongated forms and tended to align in parallel cords. Inununofluorescence showed that strong CD 105 staining was found in adhesion sites in CD 105 transfectants. Sequential observation of the growth characteristics of CD 105 transfectants and wound healing assays showed directional movement. CD 105 overexpression led to cell spreading in serum-free DMEM medium. It was blocked by a RGD containing peptide but not a RAD one. The anti-CD 105 mAb, E9, increased spreading of CD 105 transfectants, which was blocked by a RGD containing peptide but not a RAD one. These data suggest that CD 105 may change cell morphology through interaction of its RGD in the extra-cellular domain with integrins. In summary, a hypothesis was proposed that CD 105 mediated a transition of TGF-beta1 from anti-angiogenesis to pro-angiogenesis by inhibiting ALK5(T?RI)/Smad3 leading to a relative increase in ALK1/Smad5 signalling. This obviously requires further work for its validation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available