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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

Mutant IGFBP-5 is pro-fibrotic ex vivo in human skin.Human skin from four different donors was injected with the indicated IGFBP-5 expressing adenoviruses and maintained in organ culture for 10 days. (A) Skin was harvested and examined histologically. (B) Dermal thickness was measured in human skin explants. (C) ECM was quantified using hydroxyproline assay. Data in (B) and (C) represent mean and standard deviation.
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pone.0130546.g003: Mutant IGFBP-5 is pro-fibrotic ex vivo in human skin.Human skin from four different donors was injected with the indicated IGFBP-5 expressing adenoviruses and maintained in organ culture for 10 days. (A) Skin was harvested and examined histologically. (B) Dermal thickness was measured in human skin explants. (C) ECM was quantified using hydroxyproline assay. Data in (B) and (C) represent mean and standard deviation.

Mentions: To extend the in vitro findings, we engineered human skin maintained in organ culture to express wild type IGFBP-5, the NLS mutant, and the IGF-binding mutant. All three proteins exerted similar pro-fibrotic effects in that they induced dermal thickness in human skin, as measured on H&E-stained sections of paraffin-embedded skin samples (Fig 3A and 3B). The increase in dermal thickness paralleled increased hydroxyproline levels in skin from four different donors (Fig 3C). The ex vivo results further confirm that mutating the NLS or IGF-binding domains of IGFBP-5 does not impact its pro-fibrotic effect.


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)

Mutant IGFBP-5 is pro-fibrotic ex vivo in human skin.Human skin from four different donors was injected with the indicated IGFBP-5 expressing adenoviruses and maintained in organ culture for 10 days. (A) Skin was harvested and examined histologically. (B) Dermal thickness was measured in human skin explants. (C) ECM was quantified using hydroxyproline assay. Data in (B) and (C) represent mean and standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130546.g003: Mutant IGFBP-5 is pro-fibrotic ex vivo in human skin.Human skin from four different donors was injected with the indicated IGFBP-5 expressing adenoviruses and maintained in organ culture for 10 days. (A) Skin was harvested and examined histologically. (B) Dermal thickness was measured in human skin explants. (C) ECM was quantified using hydroxyproline assay. Data in (B) and (C) represent mean and standard deviation.
Mentions: To extend the in vitro findings, we engineered human skin maintained in organ culture to express wild type IGFBP-5, the NLS mutant, and the IGF-binding mutant. All three proteins exerted similar pro-fibrotic effects in that they induced dermal thickness in human skin, as measured on H&E-stained sections of paraffin-embedded skin samples (Fig 3A and 3B). The increase in dermal thickness paralleled increased hydroxyproline levels in skin from four different donors (Fig 3C). The ex vivo results further confirm that mutating the NLS or IGF-binding domains of IGFBP-5 does not impact its pro-fibrotic effect.

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