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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

Mutating the NLS domain of IGFBP-5 reduces its ability to bind nucleolin.The interaction of Flag-tagged IGFBP-5 and nucleolin was detected using immunoprecipitation using anti-Flag antibody followed by immunoblotting to detect nucleolin. Input nucleolin was detected in lysates prior to co-precipitation to ensure that samples contained equivalent amounts of protein.
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pone.0130546.g006: Mutating the NLS domain of IGFBP-5 reduces its ability to bind nucleolin.The interaction of Flag-tagged IGFBP-5 and nucleolin was detected using immunoprecipitation using anti-Flag antibody followed by immunoblotting to detect nucleolin. Input nucleolin was detected in lysates prior to co-precipitation to ensure that samples contained equivalent amounts of protein.

Mentions: To determine if the pro-fibrotic effects of IGFBP-5 require nucleolin, we silenced nucleolin in primary human fibroblasts and expressed wild-type and mutant IGFBP-5. Efficient silencing of nucleolin using sequence-specific siRNA was observed as nucleolin protein levels following silencing decreased by 65% compared to the control siRNA (Fig 5A). Silencing nucleolin did not change levels of secreted IGFBP-5 (Fig 5A) but reduced IGFBP-5 translocation to the nucleus (Fig 5B). These findings suggest that IGFBP-5 translocation to the nucleus is dependent on nucleolin. However, silencing nucleolin did not block the ability of IGFBP-5 to induce ECM production (Fig 5C). Co-immunoprecipitation of wild type and NLS mutant IGFBP-5 showed decreased interaction of the NLS mutant with nucleolin (Fig 6). These findings suggest that IGFBP-5 may interact with nucleolin via the NLS domain, and that its interaction with nucleolin may be necessary for the translocation of IGFBP-5 to the nucleus. Since mutating the NLS of IGFBP-5 did not impact its ECM-inducing effects, it is not surprising that reducing IGFBP-5 translocation to the nucleus by silencing nucleolin also did not abrogate the ECM-promoting effects of IGFBP-5.


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)

Mutating the NLS domain of IGFBP-5 reduces its ability to bind nucleolin.The interaction of Flag-tagged IGFBP-5 and nucleolin was detected using immunoprecipitation using anti-Flag antibody followed by immunoblotting to detect nucleolin. Input nucleolin was detected in lysates prior to co-precipitation to ensure that samples contained equivalent amounts of protein.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130546.g006: Mutating the NLS domain of IGFBP-5 reduces its ability to bind nucleolin.The interaction of Flag-tagged IGFBP-5 and nucleolin was detected using immunoprecipitation using anti-Flag antibody followed by immunoblotting to detect nucleolin. Input nucleolin was detected in lysates prior to co-precipitation to ensure that samples contained equivalent amounts of protein.
Mentions: To determine if the pro-fibrotic effects of IGFBP-5 require nucleolin, we silenced nucleolin in primary human fibroblasts and expressed wild-type and mutant IGFBP-5. Efficient silencing of nucleolin using sequence-specific siRNA was observed as nucleolin protein levels following silencing decreased by 65% compared to the control siRNA (Fig 5A). Silencing nucleolin did not change levels of secreted IGFBP-5 (Fig 5A) but reduced IGFBP-5 translocation to the nucleus (Fig 5B). These findings suggest that IGFBP-5 translocation to the nucleus is dependent on nucleolin. However, silencing nucleolin did not block the ability of IGFBP-5 to induce ECM production (Fig 5C). Co-immunoprecipitation of wild type and NLS mutant IGFBP-5 showed decreased interaction of the NLS mutant with nucleolin (Fig 6). These findings suggest that IGFBP-5 may interact with nucleolin via the NLS domain, and that its interaction with nucleolin may be necessary for the translocation of IGFBP-5 to the nucleus. Since mutating the NLS of IGFBP-5 did not impact its ECM-inducing effects, it is not surprising that reducing IGFBP-5 translocation to the nucleus by silencing nucleolin also did not abrogate the ECM-promoting effects of IGFBP-5.

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