Identification of Paired-related Homeobox Protein 1 as a key mesenchymal transcription factor in pulmonary fibrosis
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This manuscript will be of interest to scientists in the field of tissue injury and repair. It provides novel molecular mechanisms of a transcription factor, Prrx1, in fibroblast activation following lung injury. Overall, the work suggests that PRRX1 plays a functional role downstream of TGFb1 to elicit some aspects of the fibrotic response and that PRRX1 could represent an important therapeutic target to treat fibrosis. The strengths of this work are the multiple approaches applying human and mouse lung tissue used by the authors to test the role of PRRX1 in lung fibrosis, however, in its current form, major limitations need to be addressed.
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Abstract
Matrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF). Targeting cells driving matrix remodeling could be a promising avenue for IPF treatment. Analysis of transcriptomic database identified the mesenchymal transcription factor PRRX1 as upregulated in IPF. PRRX1, strongly expressed by lung fibroblasts, was regulated by a TGF-β/PGE2 balance in vitro in control and IPF human lung fibroblasts, while IPF fibroblast-derived matrix increased PRRX1 expression in a PDGFR-dependent manner in control ones. PRRX1 inhibition decreased human lung fibroblast proliferation by downregulating the expression of S phase cyclins. PRRX1 inhibition also impacted TGF-β driven myofibroblastic differentiation by inhibiting SMAD2/3 phosphorylation through phosphatase PPM1A upregulation and TGFBR2 downregulation, leading to TGF-β response global decrease. Finally, targeted inhibition of Prrx1 attenuated fibrotic remodeling in vivo with intra-tracheal antisense oligonucleotides in bleomycin mouse model of lung fibrosis and ex vivo using human and mouse precision-cut lung slices. Our results identified PRRX1 as a key mesenchymal transcription factor during lung fibrogenesis.
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Author Response
Reviewer #1 (Public Review):
Marchal-Duval et al studied the role of Prrx1 in lung fibroblasts. Prrx1 is a transcription factor expressed in lung fibroblasts but not in other cell types. The authors showed that Prrx1 gene expression was enhanced in IPF patients. Immunohistochemistry in IPF tissue suggested that Prrx1 was expressed in fibroblasts in fibroblastic foci. The authors then showed that Prrx1 expression was regulated by TGF-b1 stimulation or stiffness of substrate by in vitro experiments using primary human lung fibroblasts from either normal or IPF lungs. The authors also showed that Prrx1 regulated fibroblast proliferation and TGF-b signaling by regulating PPM1A and Tgfbr2 expression. Finally, the authors revealed that Prrx1 knockdown suppressed fibrosis in bleomycin-induced fibrosis or PCLS. This …
Author Response
Reviewer #1 (Public Review):
Marchal-Duval et al studied the role of Prrx1 in lung fibroblasts. Prrx1 is a transcription factor expressed in lung fibroblasts but not in other cell types. The authors showed that Prrx1 gene expression was enhanced in IPF patients. Immunohistochemistry in IPF tissue suggested that Prrx1 was expressed in fibroblasts in fibroblastic foci. The authors then showed that Prrx1 expression was regulated by TGF-b1 stimulation or stiffness of substrate by in vitro experiments using primary human lung fibroblasts from either normal or IPF lungs. The authors also showed that Prrx1 regulated fibroblast proliferation and TGF-b signaling by regulating PPM1A and Tgfbr2 expression. Finally, the authors revealed that Prrx1 knockdown suppressed fibrosis in bleomycin-induced fibrosis or PCLS. This manuscript identified novel molecular roles of Prrx1 in fibroblast activation, which is expressed in not only lung fibroblasts but also in other injured or developing organs. To support the idea that Prrx1 plays a critical role in lung fibrosis, however, some discrepancies between in vitro and in vivo data need to be clarified.
Comment #1. Although the authors showed that Prrx1 knockdown in primary fibroblasts reduced Smad2/3 phosphorylation, the reduction of Acta2 or Col1a1 after Prrx1 knockdown and TGF-b1 stimulation was not impressive (Fig. S6), suggesting that the inhibition of TGF-b signaling by Prrx1 knockdown is only partial. In contrast, Prrx1 knockdown by ASO in bleomycin-induced fibrosis showed remarkable fibrosis suppression (Fig. 6, 7). Admittedly there are differences in models and nucleotides used, but this discrepancy needs to be addressed.
We agree with the reviewer that Prrx1 inhibition only partially affects the upregulation of ACTA2, but this effect was significant (around 50% inhibition at the protein level). As stated in the discussion (lines 569-572), our data show that key ECM proteins such as Collagen 1 and Fibronectin were still upregulated in TGF-1 stimulated lung fibroblasts transfected with PRRX1 siRNA, whereas TNC and ELN mRNA expression levels were perturbed. These findings suggest that broader phenotypical changes are associated with Prrx1 knockdown. Notably, we also observed that Prrx1 inhibition impacted cell proliferation in vitro. We believe that the observed suppression of fibrosis in bleomycin treated mice following Prrx1 knockdown by ASO is the result of both the partial inhibition of TGF-β1 effect and the decrease in mesenchymal cell proliferation. Supporting this hypothesis, we observed a decrease in PDGFR-positive cell proliferation in Prrx1 ASO-treated animals (see comment #4 hereafter).
Comment #2. Fig.6 and 7 lack control groups, where mice are treated with PBS instead of bleomycin and treated with either control ASO or Prrx1 ASO.
As stated in the revised version of the material and method (line 683-686), the knockdown efficiency of Prrx1 ASO and lack of effects of control ASO were first validated in naive mice, which were treated with either Prrx1 ASO or control ASO, compared to PBS-treated mice (see Figure R2 in the answer to comment #11 of reviewer 2). Those groups were not repeated / included in the first set of bleomycin experiments in order to comply with institutional regulation to limit animal usage. In the first set of experiments (Prrx1 ASO treatment between day 7 and day 13 after bleomycin insult), the saline + PBS was just used to confirm fibrosis development while the bleomycin + Control ASO was the proper control of the bleomycin + Prrx1 ASO group. In the new second set of experiment (ASO treatment between day 21 and day28 suggested by reviewer #2), we were authorized by our local animal ethical committee to include a control ASO group in the saline treated group to confirm that the lack of effect of these control ASO compared to the PBS group (see new Figure 7-figure supplement 1).
Comment #3. In Fig. 6F, the hydroxyproline content is shown with ug collagen/ug protein. Total protein in the lung is influenced by infiltration of hematopoietic cells, which are the major population in injured lungs by cell count. Fibrosis should be ideally assessed as ug hydroxyproline/lung (or lobe).
We completely agree with the reviewer that hydroxyproline content should ideally be assessed by lobe/lung. As stated in the revised material and methods (lines 882-885), hydroxyproline and protein contents were measured using paraffin lung sections (15 sections of 10µm per sample) with the Quickzyme Biosciences hydroxyproline assay and total protein assay kits; due to limited material access and to refine its use to limit animal usage. Furthermore, the infiltration of hematopoietic cells would rather undermine the effect of Prrx1 ASO (less fibrosis and inflammation) since the contribution of those cells would be higher in control ASO-treated bleomycin mice. Considering the reviewer’s concern, a complete lobe was used to measure hydroxyproline content in the new set of experiments generated during the revision of the manuscript (see new Figure 7-figure supplement 1).
Comment #4. Major proliferating populations in bleomycin-treated lungs are not mesenchymal cells but epithelial/endothelial/hematopoietic cells. Mki67+ cells (Fig. 7D) need to be identified by co-staining with mesenchymal markers if the authors claim that Prrx1 knockdown suppresses fibroblast proliferation in vivo.
We agree with the reviewer that epithelial/endothelial/hematopoietic cells are the main proliferating populations in bleomycin treated animals at day 14. As suggested by the reviewer, we performed a MKI67 / PDGFR co-staining to identify proliferating mesenchymal cells and confirmed a decrease in proliferation in these cells after Prrx1 knock down in bleomycin treated mice (see lines 448-451 and Figure 6-figure supplement 3).
Comment #5 Bleomycin-injured lungs or IPF tissue are patchy and mixed with normal and abnormal areas. Therefore, how areas of interest are chosen for histological quantifications (Fig. 6C, S14D) need to be described in the methods section.
As now stated in the revised material section (lines 864-866), areas of interest were chosen according to the presence of major alveolar thickening as well as fibrous changes and masses (confirmed by picrosirius staining on serial section).
Reviewer #2 (Public Review):
The paper from Marchal-Duval et al reports for the first time the important role played by the transcription factor PRRX1, expressed specifically in the mesenchyme of the lung, in the context of fibrosis. The authors used a combination of human (Donor and IPF) and mouse lungs (saline and bleomycin treated) as well as associated fibroblasts and PCLS to test the functional role of PRRX1 in the context of proliferation and differentiation induced by TGFb1. The work is supported by an impressive amount of data (7 main figures and 14 supplementary figures).
Comment #1: A main weakness in this work is the counterintuitive result that PRRX1 is downregulated in human lung fibroblasts (from both IPF and Donor) treated with TGFb1.
We agree with reviewer that PRRX1 downregulation upon TGFb1 treatment may appear counterintuitive. First, as stated in the manuscript, this inhibitory effect is partial. Secondly, we performed additional experiments in the revised manuscript to better understand (timewise) the downregulation of PRRX1 in response to TGF-b1 in lung fibroblast as suggested by the reviewer. Time course analysis of PRRX1 isoform expression levels showed that PRRX1 was downregulated only after 48h. This late downregulation of PRRX1 in response to TGF-b1, could be the signature of a negative feedback loop to limit cell-responsiveness to TGF-b1 when lung fibroblasts are fully differentiated into myofibroblasts at 48h as discussed in the revised manuscript (see lines 175-180 and lines 589-594).
Comment #2: Another smaller weakness is the inactivation of Prrx1 in vivo using ASO starting at d7 post bleomycin treatment.
In our study of Prrx1 inhibition in vivo, we followed a therapeutic/interventional protocol consistent with current literature on the bleomycin model of lung fibrosis (Moeller A. et al, Int J Biochem Cell Biol 2008 and Kolb M. et al., Eur Resp J. 2020), treating the animals with either control or Prrx1 ASO every other day between day 7 and day 14 during the active fibrotic phase. In the revised manuscript, we extended our investigation to assess the potential effect of Prrx1 inhibition during the late fibrosis phase after bleomycin treatment at day 28, treating the animals with either control or Prrx1 ASO every other day between day 21 and day 27. Interestingly, we found that the effects of Prrx1 inhibition during the late fibrosis phase were less (but still) potent compared to the active fibrotic phase (see Figure 7-figure supplement 1).
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eLife assessment
This manuscript will be of interest to scientists in the field of tissue injury and repair. It provides novel molecular mechanisms of a transcription factor, Prrx1, in fibroblast activation following lung injury. Overall, the work suggests that PRRX1 plays a functional role downstream of TGFb1 to elicit some aspects of the fibrotic response and that PRRX1 could represent an important therapeutic target to treat fibrosis. The strengths of this work are the multiple approaches applying human and mouse lung tissue used by the authors to test the role of PRRX1 in lung fibrosis, however, in its current form, major limitations need to be addressed.
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Reviewer #1 (Public Review):
Marchal-Duval et al studied the role of Prrx1 in lung fibroblasts. Prrx1 is a transcription factor expressed in lung fibroblasts but not in other cell types. The authors showed that Prrx1 gene expression was enhanced in IPF patients. Immunohistochemistry in IPF tissue suggested that Prrx1 was expressed in fibroblasts in fibroblastic foci. The authors then showed that Prrx1 expression was regulated by TGF-b1 stimulation or stiffness of substrate by in vitro experiments using primary human lung fibroblasts from either normal or IPF lungs. The authors also showed that Prrx1 regulated fibroblast proliferation and TGF-b signaling by regulating PPM1A and Tgfbr2 expression. Finally, the authors revealed that Prrx1 knockdown suppressed fibrosis in bleomycin-induced fibrosis or PCLS. This manuscript identified novel …
Reviewer #1 (Public Review):
Marchal-Duval et al studied the role of Prrx1 in lung fibroblasts. Prrx1 is a transcription factor expressed in lung fibroblasts but not in other cell types. The authors showed that Prrx1 gene expression was enhanced in IPF patients. Immunohistochemistry in IPF tissue suggested that Prrx1 was expressed in fibroblasts in fibroblastic foci. The authors then showed that Prrx1 expression was regulated by TGF-b1 stimulation or stiffness of substrate by in vitro experiments using primary human lung fibroblasts from either normal or IPF lungs. The authors also showed that Prrx1 regulated fibroblast proliferation and TGF-b signaling by regulating PPM1A and Tgfbr2 expression. Finally, the authors revealed that Prrx1 knockdown suppressed fibrosis in bleomycin-induced fibrosis or PCLS. This manuscript identified novel molecular roles of Prrx1 in fibroblast activation, which is expressed in not only lung fibroblasts but also in other injured or developing organs. To support the idea that Prrx1 plays a critical role in lung fibrosis, however, some discrepancies between in vitro and in vivo data need to be clarified.
1. Although the authors showed that Prrx1 knockdown in primary fibroblasts reduced Smad2/3 phosphorylation, the reduction of Acta2 or Col1a1 after Prrx1 knockdown and TGF-b1 stimulation was not impressive (Fig. S6), suggesting that the inhibition of TGF-b signaling by Prrx1 knockdown is only partial. In contrast, Prrx1 knockdown by ASO in bleomycin-induced fibrosis showed remarkable fibrosis suppression (Fig. 6, 7). Admittedly there are differences in models and nucleotides used, but this discrepancy needs to be addressed.
2. Fig.6 and 7 lack control groups, where mice are treated with PBS instead of bleomycin and treated with either control ASO or Prrx1 ASO.
3. In Fig. 6F, the hydroxyproline content is shown with ug collagen/ug protein. Total protein in the lung is influenced by infiltration of hematopoietic cells, which are the major population in injured lungs by cell count. Fibrosis should be ideally assessed as ug hydroxyproline/lung (or lobe).
4. Major proliferating populations in bleomycin-treated lungs are not mesenchymal cells but epithelial/endothelial/hematopoietic cells. Mki67+ cells (Fig. 7D) need to be identified by co-staining with mesenchymal markers if the authors claim that Prrx1 knockdown suppresses fibroblast proliferation in vivo.
5. Bleomycin-injured lungs or IPF tissue are patchy and mixed with normal and abnormal areas. Therefore, how areas of interest are chosen for histological quantifications (Fig. 6C, S14D) need to be described in the methods section.
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Reviewer #2 (Public Review):
The paper from Marchal-Duval et al reports for the first time the important role played by the transcription factor PRRX1, expressed specifically in the mesenchyme of the lung, in the context of fibrosis. The authors used a combination of human (Donor and IPF) and mouse lungs (saline and bleomycin treated) as well as associated fibroblasts and PCLS to test the functional role of PRRX1 in the context of proliferation and differentiation induced by TGFb1. The work is supported by an impressive amount of data (7 main figures and 14 supplementary figures).
A main weakness in this work is the counterintuitive result that PRRX1 is downregulated in human lung fibroblasts (from both IPF and Donor) treated with TGFb1. Another smaller weakness is the inactivation of Prrx1 in vivo using ASO starting at d7 post …
Reviewer #2 (Public Review):
The paper from Marchal-Duval et al reports for the first time the important role played by the transcription factor PRRX1, expressed specifically in the mesenchyme of the lung, in the context of fibrosis. The authors used a combination of human (Donor and IPF) and mouse lungs (saline and bleomycin treated) as well as associated fibroblasts and PCLS to test the functional role of PRRX1 in the context of proliferation and differentiation induced by TGFb1. The work is supported by an impressive amount of data (7 main figures and 14 supplementary figures).
A main weakness in this work is the counterintuitive result that PRRX1 is downregulated in human lung fibroblasts (from both IPF and Donor) treated with TGFb1. Another smaller weakness is the inactivation of Prrx1 in vivo using ASO starting at d7 post bleomycin treatment.
The strengths of this work are the multiple approaches used by the authors to test the role of PRRX1 in lung fibrosis. The results are statistically solid and informative. The results presented are extremely convincing to support their conclusion that PRRX1, downstream of TGFb1 signaling is important for fibrosis.
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