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. 2022 Feb;66(2):146-157.
doi: 10.1165/rcmb.2021-0022OC.

Regulator of Cell Cycle Protein (RGCC/RGC-32) Protects against Pulmonary Fibrosis

Irina G Luzina  1   2 , Violeta Rus  1   2 , Virginia Lockatell  1   2 , Jean-Paul Courneya  3 , Brian S Hampton  1 , Rita Fishelevich  1   2 , Alexander V Misharin  4 , Nevins W Todd  1   2 , Tudor C Badea  5   6 , Horea Rus  1   2 , Sergei P Atamas  1   2
Affiliations

Regulator of Cell Cycle Protein (RGCC/RGC-32) Protects against Pulmonary Fibrosis

Irina G Luzina et al. Am J Respir Cell Mol Biol. 2022 Feb.

Abstract

Some previous studies in tissue fibrosis have suggested a profibrotic contribution from elevated expression of a protein termed either RGCC (regulator of cell cycle) or RGC-32 (response gene to complement 32 protein). Our analysis of public gene expression datasets, by contrast, revealed a consistent decrease in RGCC mRNA levels in association with pulmonary fibrosis. Consistent with this observation, we found that stimulating primary adult human lung fibroblasts with transforming growth factor (TGF)-β in cell cultures elevated collagen expression and simultaneously attenuated RGCC mRNA and protein levels. Moreover, overexpression of RGCC in cultured lung fibroblasts attenuated the stimulating effect of TGF-β on collagen levels. Similar to humans with pulmonary fibrosis, the levels of RGCC were also decreased in vivo in lung tissues of wild-type mice challenged with bleomycin in both acute and chronic models. Mice with constitutive RGCC gene deletion accumulated more collagen in their lungs in response to chronic bleomycin challenge than did wild-type mice. RNA-Seq analyses of lung fibroblasts revealed that RGCC overexpression alone had a modest transcriptomic effect, but in combination with TGF-β stimulation, induced notable transcriptomic changes that negated the effects of TGF-β, including on extracellular matrix-related genes. At the level of intracellular signaling, RGCC overexpression delayed early TGF-β-induced Smad2/3 phosphorylation, elevated the expression of total and phosphorylated antifibrotic mediator STAT1, and attenuated the expression of a profibrotic mediator STAT3. We conclude that RGCC plays a protective role in pulmonary fibrosis and that its decline permits collagen accumulation. Restoration of RGCC expression may have therapeutic potential in pulmonary fibrosis.

Keywords: idiopathic pulmonary fibrosis; lung fibrosis; regulator of cell cycle protein; response gene to complement 32 protein; scleroderma lung disease.

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Figures

Figure 1.
Figure 1.
Expression levels of RGCC (regulator of cell cycle) in the lungs of patients with interstitial lung disease (ILD) and healthy controls. (A) Phylogenetic tree of RGCC proteins by species. Percent similarities with human RGCC are indicated. (B) Normalized DESeq2 counts from bulk RNA-Seq of lung samples from macroscopically normal-appearing and scarred areas of with idiopathic pulmonary fibrosis (IPF) lungs (IPFn and IPFs, respectively) as well as from HC (24). The indicated P value was calculated using the DESeq2 likelihood ratio tests. (C) RT-qPCR for RGCC mRNA normalized to 18S rRNA levels and further normalized to the average of the control group; in the ILD group, tissue samples from patients with IPF are denoted with triangles whereas the SSc-ILD tissue samples are denoted with squares. The indicated P value was calculated using Mann-Whitney U test. (D) Violin plot of RGCC expression in pulmonary cell population clusters (25). (E) Split dot-plots of RGCC expression in the indicated pulmonary cell types. Blue dots denote HC, whereas red dots indicate patients with ILD described in (25). (F) Violin plots of RGCC expression in the fibroblast cluster control and ILD lungs (25). (G) Western blotting for RGCC in lung homogenates obtained from healthy controls (Ctrl) and patients with IPF. The ratios of RGCC band densities to their corresponding GAPDH bands are indicated above the gels. AT1 = alveolar type 1; HC = healthy controls; IPF = idiopathic pulmonary fibrosis; Max = maximum; Min = minimum; NKT = natural killer T; PM = polymyositis; SSc = systemic sclerosis.
Figure 2.
Figure 2.
RGCC expression in primary lung fibroblast cultures. (A) Western blotting of primary fibroblast lysates for COL1, α-SMA, RGCC, and β-actin. The ratios of COL1, α-SMA, and RGCC band densities to their corresponding β-actin bands as well as statistical significance of differences between normal human lung fibroblast (NHLF) and IPF fibroblasts are shown in the scatterplots on the right. (B) RT-qPCR for collagen I (α2) and RGCC mRNA in NHLFs and IPF lung fibroblast cultures that were or were not stimulated with 5 ng/ml of TGF-β. Asterisk indicates significant differences (*P < 0.05) in triplicate TGF-β–stimulated versus control cultures for each condition. (C) Western blotting of primary fibroblast lysates for RGCC and GAPDH. NHLF and IPF lung fibroblast cultures were or were not stimulated with 5 ng/ml of TGF-β. The ratios of RGCC band densities to their corresponding GAPDH bands are indicated above the gel. (D) Effect of plasmid-based RGCC gene delivery to NHLFs and IPF lung fibroblasts on TGF-β–activated type I collagen protein expression. Western blots for type I collagen, GAPDH, and RGCC are shown. The ratios of collagen band densities to their corresponding GAPDH bands are indicated above the gels, and the ratios of RGCC bands to GAPDH are shown next to the indications of the plasmids used for cell transfections below the gels. α-SMA = α-smooth muscle actin; COL1 = collagen type 1; OD = optical density; TGF = transforming growth factor.
Figure 3.
Figure 3.
Pulmonary changes induced in WT and RGCC KO mice by acute or chronic bleomycin (BLM) challenges in comparison to similarly challenged with PBS control animals. (A) RT-qPCR results for RGCC mRNA in the acute bleomycin injury model. (B) Western blotting for RGCC and GAPDH in lung homogenates obtained from bleomycin-challenged mice in the acute and chronic bleomycin injury models. (C) Total lung collagen, microgram per milligram of wet lung tissue, after acute bleomycin challenge. (D) Time-dependent changes in total body weight of mice in the chronic bleomycin model. Asterisk indicates significant differences (*P < 0.05) between BLM-treated and PBS-treated mice. (E) Trichrome staining of lung sections from mice in the chronic bleomycin model imaged at lower (top row) and higher (bottom row) magnifications. The framed areas of the lower-magnification images are shown at a higher magnification in the bottom row. Scale bars (left side of each row), 200 μm. Selected areas of plural thickening are indicated with arrows, and collagen fibers appearing in blue are indicated with arrowheads. (F) Total and differential cell counts in bronchoalveolar lavage samples from mouse lungs in the chronic bleomycin model. Asterisk indicates significant increases (*P < 0.05) in bleomycin-challenged animals compared with PBS controls. (G) Total lung collagen, microgram per milligram of wet lung tissue, after chronic bleomycin challenge. In A, C, and G, the numbers of animals tested in each group (n) are shown, as well as P values calculated using a two-tailed Student’s t test for the indicated pairwise comparisons. In D, E, and F, data represent 4–5 animals tested for each experimental condition. Gran = granulocytes; KO = knockout; Lymph = lymphocytes; Mϕ = macrophages; WT = wild-type.
Figure 4.
Figure 4.
Transcriptomic effects of RGCC overexpression in NHLFs. (A) Volcano plots [–log10(P value) versus log2(fold)] of genes in the indicated comparisons. Selection criteria for up- and downregulated genes included significant differences (P < 0.05) as well as fold-difference between groups. The total numbers of genes meeting the selection criteria for elevated (orange for > 10-fold increase and green for 2–10-fold increase) or reduced (purple for > 10-fold decrease and blue for 2–10-fold decrease) expression are indicated in each panel. (B) Pathway and process enrichment analyses of differentially expressed genes were performed in Metascape. Clustering of the resulting top enriched terms and of the indicated pairwise comparisons is represented as the heatmap. The colors denote the negative log10 of P values for pathway/process enrichment. (C) Metascape-generated Circos visualization of overlaps among gene lists corresponding to indicated pairwise comparisons. Gene pairs falling into the same enrichment term linkage are represented with blue curves. (D) Changes in the expression levels of selected indicated extracellular matrix-related genes induced by TGF-β stimulation (red) or RGCC overexpression in TGF-β–stimulated cells (blue). The elevated expression in response to stimulation with TGF-β is indicated as log2 fold difference from NULL-transfected cells without additional stimulation. The downregulated gene expression in RGCC-overexpressing cells is shown as log2 fold difference from NULL-transfected and additionally TGF-β–treated cells.
Figure 5.
Figure 5.
Effect of RGCC overexpression on selected intracellular signaling mediators. (A) Smad2/3 phosphorylation, after stimulation with rhTGF-β for indicated times, in NHLF1 transfected with either noncoding NULL or RGCC-encoding plasmids. The ratios of phosphorylated to total Smad2/3 densities are indicated above the corresponding Western blotting gel lanes. (B) Western blotting for indicated intracellular signaling mediators in NHLF transfected with the RGCC-encoding or control NULL plasmids.
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