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IGFBP-5 Promotes Fibrosis Independently of Its Translocation to the Nucleus and Its Interaction with Nucleolin and IGF.

Su Y, Nishimoto T, Feghali-Bostwick C - PLoS ONE (2015)

Bottom Line: We examined the effect of nucleolin on IGFBP-5 localization and function via sequence-specific silencing in primary human fibroblasts.Silencing nucleolin reduced IGFBP-5 translocation to the nucleus but did not block the ability of IGFBP-5 to induce ECM production and a fibrotic phenotype.However, nuclear translocation is not necessary for IGFBP-5 fibrotic activity; neither is IGF binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Rheumatology & Immunology, Medical University of South Carolina, Charleston, South Carolina, United States of America.

ABSTRACT

Background: Insulin-like growth factor binding protein (IGFBP)-5 levels are increased in systemic sclerosis (SSc) skin and lung. We previously reported that IGFBP-5 is a pro-fibrotic factor that induces extracellular matrix (ECM) production and deposition. 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 or a mutated IGF binding site. Abrogation of nuclear translocation in the NLS mutant was confirmed using immunofluorescence and immunoblotting of nuclear and cytoplasmic cellular extracts. Abrogation of IGF binding was confirmed using western ligand blot. The fibrotic activity of wild type and 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. We examined the effect of nucleolin 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. The NLS-mutant also induced fibrosis ex vivo in human skin, thus confirming and extending the in vitro findings. Similar findings were obtained with the IGF-binding mutant. Nucleolin, a nucleolar protein that can serve as a nuclear receptor, was identified as an IGFBP-5 binding partner. Silencing nucleolin reduced IGFBP-5 translocation to the nucleus but did not block the ability of IGFBP-5 to induce ECM production and a fibrotic phenotype.

Conclusions: IGFBP-5 transport to the nucleus requires an intact NLS and nucleolin. However, nuclear translocation is not necessary for IGFBP-5 fibrotic activity; neither is IGF binding. Our data provide further insights into the role of cellular compartmentalization in IGFBP-5-induced fibrosis.

No MeSH data available.


Related in: MedlinePlus

Generation of IGFBP-5 with mutated IGF binding and NLS domains and validation of the loss of function imparted by the mutations.(A) Schematic of wild type, IGF binding site-mutant, and NLS-mutant IGFBP-5 expressing constructs used for the generation of adenoviruses. (B) Confirmation that all three constructs result in IGFBP-5 production by western blotting and validation of the loss of binding to IGF of the IGF-binding site mutant using western ligand blotting. (C) The NLS-mutant IGFBP-5 does not translocate to the nucleus of primary human fibroblasts as detected using immunofluorescence. (D) The NLS-mutant IGFBP-5 is not detected in nuclear extracts of primary human fibroblasts expressing the indicated constructs, but its levels were higher in cytoplasmic fractions as detected following cellular fractionation and immunoblotting.
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pone.0130546.g001: Generation of IGFBP-5 with mutated IGF binding and NLS domains and validation of the loss of function imparted by the mutations.(A) Schematic of wild type, IGF binding site-mutant, and NLS-mutant IGFBP-5 expressing constructs used for the generation of adenoviruses. (B) Confirmation that all three constructs result in IGFBP-5 production by western blotting and validation of the loss of binding to IGF of the IGF-binding site mutant using western ligand blotting. (C) The NLS-mutant IGFBP-5 does not translocate to the nucleus of primary human fibroblasts as detected using immunofluorescence. (D) The NLS-mutant IGFBP-5 is not detected in nuclear extracts of primary human fibroblasts expressing the indicated constructs, but its levels were higher in cytoplasmic fractions as detected following cellular fractionation and immunoblotting.

Mentions: The domains of IGFBP-5 that bind to IGF-I localize to the N-terminal domain at aa49-74 and C-terminal domain at aa208-218, with the N-terminal domain being the high affinity binding site while the C-terminal region contains a low affinity binding site. This latter site overlaps with the ECM binding site and the NLS. To delineate the role of the NLS and IGF-binding domains in fibrosis, we generated three IGFBP-5 constructs: a wild type human IGFBP-5 [2] to which we added a FLAG tag, IGFBP-5 with a mutated NLS with aa214-aa218 mutated from RGRKR to MDGEA [8], and IGFBP-5 with a mutated IGF binding domain with aa68 to aa74 mutated from KPLHALL to NQQHAQQ [22] (Fig 1A).


IGFBP-5 Promotes Fibrosis Independently of Its Translocation to the Nucleus and Its Interaction with Nucleolin and IGF.

Su Y, Nishimoto T, Feghali-Bostwick C - PLoS ONE (2015)

Generation of IGFBP-5 with mutated IGF binding and NLS domains and validation of the loss of function imparted by the mutations.(A) Schematic of wild type, IGF binding site-mutant, and NLS-mutant IGFBP-5 expressing constructs used for the generation of adenoviruses. (B) Confirmation that all three constructs result in IGFBP-5 production by western blotting and validation of the loss of binding to IGF of the IGF-binding site mutant using western ligand blotting. (C) The NLS-mutant IGFBP-5 does not translocate to the nucleus of primary human fibroblasts as detected using immunofluorescence. (D) The NLS-mutant IGFBP-5 is not detected in nuclear extracts of primary human fibroblasts expressing the indicated constructs, but its levels were higher in cytoplasmic fractions as detected following cellular fractionation and immunoblotting.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4478026&req=5

pone.0130546.g001: Generation of IGFBP-5 with mutated IGF binding and NLS domains and validation of the loss of function imparted by the mutations.(A) Schematic of wild type, IGF binding site-mutant, and NLS-mutant IGFBP-5 expressing constructs used for the generation of adenoviruses. (B) Confirmation that all three constructs result in IGFBP-5 production by western blotting and validation of the loss of binding to IGF of the IGF-binding site mutant using western ligand blotting. (C) The NLS-mutant IGFBP-5 does not translocate to the nucleus of primary human fibroblasts as detected using immunofluorescence. (D) The NLS-mutant IGFBP-5 is not detected in nuclear extracts of primary human fibroblasts expressing the indicated constructs, but its levels were higher in cytoplasmic fractions as detected following cellular fractionation and immunoblotting.
Mentions: The domains of IGFBP-5 that bind to IGF-I localize to the N-terminal domain at aa49-74 and C-terminal domain at aa208-218, with the N-terminal domain being the high affinity binding site while the C-terminal region contains a low affinity binding site. This latter site overlaps with the ECM binding site and the NLS. To delineate the role of the NLS and IGF-binding domains in fibrosis, we generated three IGFBP-5 constructs: a wild type human IGFBP-5 [2] to which we added a FLAG tag, IGFBP-5 with a mutated NLS with aa214-aa218 mutated from RGRKR to MDGEA [8], and IGFBP-5 with a mutated IGF binding domain with aa68 to aa74 mutated from KPLHALL to NQQHAQQ [22] (Fig 1A).

Bottom Line: We examined the effect of nucleolin on IGFBP-5 localization and function via sequence-specific silencing in primary human fibroblasts.Silencing nucleolin reduced IGFBP-5 translocation to the nucleus but did not block the ability of IGFBP-5 to induce ECM production and a fibrotic phenotype.However, nuclear translocation is not necessary for IGFBP-5 fibrotic activity; neither is IGF binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Rheumatology & Immunology, Medical University of South Carolina, Charleston, South Carolina, United States of America.

ABSTRACT

Background: Insulin-like growth factor binding protein (IGFBP)-5 levels are increased in systemic sclerosis (SSc) skin and lung. We previously reported that IGFBP-5 is a pro-fibrotic factor that induces extracellular matrix (ECM) production and deposition. 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 or a mutated IGF binding site. Abrogation of nuclear translocation in the NLS mutant was confirmed using immunofluorescence and immunoblotting of nuclear and cytoplasmic cellular extracts. Abrogation of IGF binding was confirmed using western ligand blot. The fibrotic activity of wild type and 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. We examined the effect of nucleolin 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. The NLS-mutant also induced fibrosis ex vivo in human skin, thus confirming and extending the in vitro findings. Similar findings were obtained with the IGF-binding mutant. Nucleolin, a nucleolar protein that can serve as a nuclear receptor, was identified as an IGFBP-5 binding partner. Silencing nucleolin reduced IGFBP-5 translocation to the nucleus but did not block the ability of IGFBP-5 to induce ECM production and a fibrotic phenotype.

Conclusions: IGFBP-5 transport to the nucleus requires an intact NLS and nucleolin. However, nuclear translocation is not necessary for IGFBP-5 fibrotic activity; neither is IGF binding. Our data provide further insights into the role of cellular compartmentalization in IGFBP-5-induced fibrosis.

No MeSH data available.


Related in: MedlinePlus