Session Information
Session Title: Systemic Sclerosis, Fibrosing Syndromes and Raynaud's - Pathogenesis, Animal Models and Genetics
Session Type: Abstract Submissions (ACR)
Background/Purpose: Insulin-like growth factor binding protein (IGFBP)-5 is one of six IGFBPs. IGFBP-5 is the most conserved member of the family. IGFBP-5 levels are increased in systemic sclerosis (SSc) skin and lung tissues. We previously reported that IGFBP-5 is a pro-fibrotic factor that induces extracellular matrix (ECM) production and deposition. IGFBP-5 promoted a fibrotic phenotype in vitro in primary human fibroblasts, in vivo in mouse lung and skin, and ex vivo in human skin. Since IGFBP-5 contains a nuclear localization signal (NLS) that facilitates its nuclear translocation, we sought to examine the role of nuclear translocation on the fibrotic activity of IGFBP-5 and identify IGFBP-5 binding partners relevant for its nuclear compartmentalization.
Methods: We generated functional wild type IGFBP-5 and IGFBP-5 with a mutated NLS. Abrogation of nuclear translocation in the NLS mutant was confirmed using immunofluorescence and immunoblotting of nuclear and cytoplasmic cellular extracts. The fibrotic activity of wild type and NLS-mutant IGFBP-5 was examined in vitro in primary human fibroblasts and ex vivo in human skin. We identified IGFBP-5 binding partners using immunoprecipitation and Mass Spectrometry. Binding of IGFBP-5 to its partner was validated using co-immunoprecipitation and immunoblotting. We examined the effect of the partner on IGFBP-5 localization and function via sequence-specific silencing in primary human fibroblasts.
Results: Our results show that IGFBP-5-induced ECM production in vitro in primary human fibroblasts is independent of its nuclear translocation as the NLS-mutant IGFBP-5 retained fibrotic activity. The NLS-mutant IGFBP-5 also induced fibrosis ex vivo in human skin maintained in organ culture, thus confirming and extending the in vitro findings. Nucleolin, a nucleolar protein that can serve as a nuclear receptor, was identified as an IGFBP-5 binding partner. Silencing nucleolin in primary human fibroblasts reduced IGFBP-5 translocation to the nucleus but did not block the ability of IGFBP-5 to induce ECM production and a fibrotic phenotype.
Conclusion: IGFBP-5 transport to nucleus requires an intact NLS and nucleolin. However, nuclear translocation is not necessary for IGFBP-5 fibrotic activity. Our data provide further insights into the mechanism mediating the fibrotic activity of IGFBP-5 and the role of nuclear compartmentalization in IGFBP-5-induced fibrosis.
Disclosure:
Y. Su,
None;
C. Feghali-Bostwick,
None.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/translocation-of-igfbp-5-to-the-nucleus-and-its-interaction-with-nucleolin-do-not-dictate-its-fibrotic-effects/