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BARD1 mediates TGF-β signaling in pulmonary fibrosis.

André PA, Prêle CM, Vierkotten S, Carnesecchi S, Donati Y, Chambers RC, Pache JC, Crestani B, Barazzone-Argiroffo C, Königshoff M, Laurent GJ, Irminger-Finger I - Respir. Res. (2015)

Bottom Line: Previous reports have shown that BARD1 expression is upregulated in response to hypoxia and associated with TGF-β signaling, both recognized factors driving lung fibrosis.Protein and mRNA expression studies showed very low expression in healthy lung tissues, but upregulated expression of full length (FL) BARD1 and BARD1β in fibrotic tissues.Our data suggest that FL BARD1 and BARD1β might be mediators of pleiotropic effects of TGF-β.

View Article: PubMed Central - PubMed

Affiliation: Molecular Gynecology and Obstetrics Laboratory, Department of Gynecology and Obstetrics, Geneva University Hospitals, Geneva, Switzerland. Pierre-Alain.Andre@unige.ch.

ABSTRACT

Background: Idiopathic pulmonary fibrosis (IPF) is a rapid progressive fibro-proliferative disorder with poor prognosis similar to lung cancer. The pathogenesis of IPF is uncertain, but loss of epithelial cells and fibroblast proliferation are thought to be central processes. Previous reports have shown that BARD1 expression is upregulated in response to hypoxia and associated with TGF-β signaling, both recognized factors driving lung fibrosis. Differentially spliced BARD1 isoforms, in particular BARD1β, are oncogenic drivers of proliferation in cancers of various origins. We therefore hypothesized that BARD1 and/or its isoforms might play a role in lung fibrosis.

Methods: We investigated BARD1 expression as a function of TGF-β in cultured cells, in mice with experimentally induced lung fibrosis, and in lung biopsies from pulmonary fibrosis patients.

Results: FL BARD1 and BARD1β were upregulated in response to TGF-β in epithelial cells and fibroblasts in vitro and in vivo. Protein and mRNA expression studies showed very low expression in healthy lung tissues, but upregulated expression of full length (FL) BARD1 and BARD1β in fibrotic tissues.

Conclusion: Our data suggest that FL BARD1 and BARD1β might be mediators of pleiotropic effects of TGF-β. In particular BARD1β might be a driver of proliferation and of pulmonary fibrosis pathogenesis and progression and represent a target for treatment.

No MeSH data available.


Related in: MedlinePlus

TGF-β induces BARD1β overexpression and localization. a Schematic representation of cDNA structure of FL BARD1 and N-terminally truncated isoform BARD1β. Approximate locations of protein motifs RING finger (RING), ankyrin repeats (ANK), and BRCT domains (BRCT) of BARD1 are indicated. Green exons indicate protein coding open reading frame (ORF). BARD1β encodes an alternative ORF in the first exon. Arrows indicated approximate positions of epitopes reactive with the antibodies used. b Lung epithelial cells (A549) and fibroblasts (L929) were cultured in absence (NT) or presence of TGF-β1, and cell extracts were prepared after 24 and 48 h. Western blots of cell extracts were probed with anti-BARD1 BL (exon 4), BARD1β-specific P25 (alternative ORF of BARD1β), α-SMA, E-cadherin, fibronectin, and β-actin antibodies. c A549 were treated with TGF-β1 and cells were fixed after 48 h. Immunofluoresence was performed with anti-E-cadherin, fibronectin, or BARD1 BL (exon 4) antibodies. d Co-immunostaining with anti-BARD1 BL and fibronectin (Fib) antibodies. TGF-β1 modulates BARD1 localization to areas at the cell membrane (yellow arrows) and cytoplasmic vesicles (white arrows), similar to and co-staining with fibronectin. Scale bars 20 μm
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Fig2: TGF-β induces BARD1β overexpression and localization. a Schematic representation of cDNA structure of FL BARD1 and N-terminally truncated isoform BARD1β. Approximate locations of protein motifs RING finger (RING), ankyrin repeats (ANK), and BRCT domains (BRCT) of BARD1 are indicated. Green exons indicate protein coding open reading frame (ORF). BARD1β encodes an alternative ORF in the first exon. Arrows indicated approximate positions of epitopes reactive with the antibodies used. b Lung epithelial cells (A549) and fibroblasts (L929) were cultured in absence (NT) or presence of TGF-β1, and cell extracts were prepared after 24 and 48 h. Western blots of cell extracts were probed with anti-BARD1 BL (exon 4), BARD1β-specific P25 (alternative ORF of BARD1β), α-SMA, E-cadherin, fibronectin, and β-actin antibodies. c A549 were treated with TGF-β1 and cells were fixed after 48 h. Immunofluoresence was performed with anti-E-cadherin, fibronectin, or BARD1 BL (exon 4) antibodies. d Co-immunostaining with anti-BARD1 BL and fibronectin (Fib) antibodies. TGF-β1 modulates BARD1 localization to areas at the cell membrane (yellow arrows) and cytoplasmic vesicles (white arrows), similar to and co-staining with fibronectin. Scale bars 20 μm

Mentions: We have shown previously that BARD1β promotes cancer cell proliferation and induces proliferation of non-transformed fibroblasts [23, 25], we wanted to monitor the specific expression of FL BARD1 and BARD1β in response to TGF-β treatment. We probed Western blots of A549 and L929 cells cultured in the absence or presence of TGF-β for expression of FL BARD1 and BARD1β using an antibody specifically recognizing the N-terminal region of BARD1β, P25 [23, 25] (Fig. 2a). A549 cells and L929, both showed an increase of FL BARD1 expression, as well as an upregulation of BARD1β after treatment with TGF-β. The increase of FL BARD1 and BARD1β expression in A549 cells was accompanied by an increase of α-smooth muscle actin and fibronectin, as well as the decrease of E-cadherin, consistent with the well-described function of TGF-β as EMT inducer [36, 37] (Fig. 2b).Fig. 2


BARD1 mediates TGF-β signaling in pulmonary fibrosis.

André PA, Prêle CM, Vierkotten S, Carnesecchi S, Donati Y, Chambers RC, Pache JC, Crestani B, Barazzone-Argiroffo C, Königshoff M, Laurent GJ, Irminger-Finger I - Respir. Res. (2015)

TGF-β induces BARD1β overexpression and localization. a Schematic representation of cDNA structure of FL BARD1 and N-terminally truncated isoform BARD1β. Approximate locations of protein motifs RING finger (RING), ankyrin repeats (ANK), and BRCT domains (BRCT) of BARD1 are indicated. Green exons indicate protein coding open reading frame (ORF). BARD1β encodes an alternative ORF in the first exon. Arrows indicated approximate positions of epitopes reactive with the antibodies used. b Lung epithelial cells (A549) and fibroblasts (L929) were cultured in absence (NT) or presence of TGF-β1, and cell extracts were prepared after 24 and 48 h. Western blots of cell extracts were probed with anti-BARD1 BL (exon 4), BARD1β-specific P25 (alternative ORF of BARD1β), α-SMA, E-cadherin, fibronectin, and β-actin antibodies. c A549 were treated with TGF-β1 and cells were fixed after 48 h. Immunofluoresence was performed with anti-E-cadherin, fibronectin, or BARD1 BL (exon 4) antibodies. d Co-immunostaining with anti-BARD1 BL and fibronectin (Fib) antibodies. TGF-β1 modulates BARD1 localization to areas at the cell membrane (yellow arrows) and cytoplasmic vesicles (white arrows), similar to and co-staining with fibronectin. Scale bars 20 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4587901&req=5

Fig2: TGF-β induces BARD1β overexpression and localization. a Schematic representation of cDNA structure of FL BARD1 and N-terminally truncated isoform BARD1β. Approximate locations of protein motifs RING finger (RING), ankyrin repeats (ANK), and BRCT domains (BRCT) of BARD1 are indicated. Green exons indicate protein coding open reading frame (ORF). BARD1β encodes an alternative ORF in the first exon. Arrows indicated approximate positions of epitopes reactive with the antibodies used. b Lung epithelial cells (A549) and fibroblasts (L929) were cultured in absence (NT) or presence of TGF-β1, and cell extracts were prepared after 24 and 48 h. Western blots of cell extracts were probed with anti-BARD1 BL (exon 4), BARD1β-specific P25 (alternative ORF of BARD1β), α-SMA, E-cadherin, fibronectin, and β-actin antibodies. c A549 were treated with TGF-β1 and cells were fixed after 48 h. Immunofluoresence was performed with anti-E-cadherin, fibronectin, or BARD1 BL (exon 4) antibodies. d Co-immunostaining with anti-BARD1 BL and fibronectin (Fib) antibodies. TGF-β1 modulates BARD1 localization to areas at the cell membrane (yellow arrows) and cytoplasmic vesicles (white arrows), similar to and co-staining with fibronectin. Scale bars 20 μm
Mentions: We have shown previously that BARD1β promotes cancer cell proliferation and induces proliferation of non-transformed fibroblasts [23, 25], we wanted to monitor the specific expression of FL BARD1 and BARD1β in response to TGF-β treatment. We probed Western blots of A549 and L929 cells cultured in the absence or presence of TGF-β for expression of FL BARD1 and BARD1β using an antibody specifically recognizing the N-terminal region of BARD1β, P25 [23, 25] (Fig. 2a). A549 cells and L929, both showed an increase of FL BARD1 expression, as well as an upregulation of BARD1β after treatment with TGF-β. The increase of FL BARD1 and BARD1β expression in A549 cells was accompanied by an increase of α-smooth muscle actin and fibronectin, as well as the decrease of E-cadherin, consistent with the well-described function of TGF-β as EMT inducer [36, 37] (Fig. 2b).Fig. 2

Bottom Line: Previous reports have shown that BARD1 expression is upregulated in response to hypoxia and associated with TGF-β signaling, both recognized factors driving lung fibrosis.Protein and mRNA expression studies showed very low expression in healthy lung tissues, but upregulated expression of full length (FL) BARD1 and BARD1β in fibrotic tissues.Our data suggest that FL BARD1 and BARD1β might be mediators of pleiotropic effects of TGF-β.

View Article: PubMed Central - PubMed

Affiliation: Molecular Gynecology and Obstetrics Laboratory, Department of Gynecology and Obstetrics, Geneva University Hospitals, Geneva, Switzerland. Pierre-Alain.Andre@unige.ch.

ABSTRACT

Background: Idiopathic pulmonary fibrosis (IPF) is a rapid progressive fibro-proliferative disorder with poor prognosis similar to lung cancer. The pathogenesis of IPF is uncertain, but loss of epithelial cells and fibroblast proliferation are thought to be central processes. Previous reports have shown that BARD1 expression is upregulated in response to hypoxia and associated with TGF-β signaling, both recognized factors driving lung fibrosis. Differentially spliced BARD1 isoforms, in particular BARD1β, are oncogenic drivers of proliferation in cancers of various origins. We therefore hypothesized that BARD1 and/or its isoforms might play a role in lung fibrosis.

Methods: We investigated BARD1 expression as a function of TGF-β in cultured cells, in mice with experimentally induced lung fibrosis, and in lung biopsies from pulmonary fibrosis patients.

Results: FL BARD1 and BARD1β were upregulated in response to TGF-β in epithelial cells and fibroblasts in vitro and in vivo. Protein and mRNA expression studies showed very low expression in healthy lung tissues, but upregulated expression of full length (FL) BARD1 and BARD1β in fibrotic tissues.

Conclusion: Our data suggest that FL BARD1 and BARD1β might be mediators of pleiotropic effects of TGF-β. In particular BARD1β might be a driver of proliferation and of pulmonary fibrosis pathogenesis and progression and represent a target for treatment.

No MeSH data available.


Related in: MedlinePlus