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Angiogenin (Ang) is known to induce cell proliferation and inhibit apoptosis by cellular signaling pathways and its direct nuclear functions, but the mechanism of action for Ang in astrocytoma is not yet clear. Astrocytoma is the most frequent one among various neurogliomas, of which a subtype known as glioblastoma multiforme (GBM) is the most malignant brain glioma and seriously influences the life quality of the patients. The expression of Ang and Bcl-xL were detected in 28 cases of various grades of astrocytoma and 6 cases of normal human tissues by quantitative real-time PCR. The results showed that the expression of Ang and Bcl-xL positively correlated with the malignant grades. Cytological experiments indicated that Ang facilitated human glioblastoma U87MG cell proliferation and knock-down of endogenous Ang promoted cell apoptosis. Furthermore, Ang activated NF-κB pathway and entered the U87MG cell nuclei, and blocking NF-κB pathway or inhibiting Ang nuclear translocation partially suppressed Ang-induced cell proliferation. The results suggested that Ang participated in the regulation of evolution process of astrocytoma by interfering NF-κB pathway and its nucleus function. In addition, four and a half LIM domains 3 (FHL3), a novel Ang binding partner, was required for Ang-mediated HeLa cell proliferation in our previous study. We also found that knockdown of FHL3 enhanced IκBα phosphorylation and overexpression of Ang inhibited FHL3 expression in U87MG cells. Together our findings suggested that Ang could activate NF-κB pathway by regulating the expression of FHL3. In conclusion, the present study established a link between Ang and FHL3 proteins and identifies a new pathway for regulating astrocytoma progression.

Four and a half LIM domain protein 3 (FHL3) is a member of the FHL protein family that plays roles in the regulation of cell survival, cell adhesion and signal transduction. However, the mechanism of action for FHL3 is not yet clear. The aim of present study was to identify novel binding partner of FHL3 and to explore the underlying mechanism. With the use of yeast two-hybrid screening system, FHL3 was used as the bait to screen human fetal hepatic cDNA library for interacting proteins. Methionine-1X was identified as a novel FHL3 binding partner. The interaction between FHL3 and the full length MT-1X was further confirmed by yeast two-hybrid assay, co-immunoprecipitation and GST pull-down assays. Furthermore,the result demonstrated that MT-1X knockdown promoted the FHL3-induced inhibitory effect on HepG2 cells by regulating FHL3-mediated Smad signaling and involving in the modulation the expression of G2/M phase-related proteins through interaction with FHL3. These findings suggest that functional interactions between FHL3 and MT-1X may provide some clues to the mechanisms of FHL3-regulated cell proliferation.

Angiogenin (Ang) is known to induce cell proliferation and inhibit apoptosis by cellular signaling pathways and by direct nuclear functions of Ang, but the mechanism of action for Ang is not yet clear. The aim of present study was to identify novel binding partner of Ang and to explore the underlying mechanism. With the use of yeast two-hybrid screening system, Ang was used as the bait to screen human fetal hepatic cDNA library for interacting proteins. Four and a half LIM domains 3 (FHL3) was identified as a novel Ang binding partner. The interaction between Ang and the full length FHL3 was further confirmed by yeast two-hybrid assay, co-immunoprecipitation and GST pull-down assays. Furthermore, FHL3 was required for Ang-mediated HeLa cell proliferation and nuclear translocation of Ang. These findings suggest that the interaction between Ang and FHL3 may provide some clues to the mechanisms of Ang-regulated cell growth and apoptosis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Four and a half LIM domain protein 3 (FHL3) is a member of the family of LIM proteins and is involved in myogenesis, cytoskeleton reconstruction, cell growth and differentiation. The full-length FHL3 cDNA was cloned from human spleen cDNA library and inserted in a prokaryotic expression vector pBV220 and then the recombinant plasmid was transformed into E. coli JM109. The expression of the recombinant protein was induced at 42 degrees C. SDS-PAGE analysis showed that recombinant human FHL3 (rhFHL3) was mainly expressed as an inclusion body. After purification by HisTrap FF crude, the rhFHL3 was renatured by dialysis against renaturing buffer and identified by Western blot analysis using human FHL3 polyclonal antibody. The MTT assay showed that the purified rhFHL3 could inhibit HepG2 cell growth but promote the proliferation of ECV304 cells. In addition, the expression of angiogenin (Ang) gene was increased when ECV304 cells were pretreated with rhFHL3.