Significance of the p38MAPK-CRP2 axis in myofibroblastic phenotypic transition

Abstract

We have recently demonstrated that a LIM domain protein, cysteine and glycine-rich protein 2 (CSRP2 [CRP2]), plays a vital role in the functional expression of myofibroblasts and cancer-associated fibroblasts. CRP2 binds directly to myocardin-related transcription factors (MRTF [MRTF-A or MRTF-B]) and serum response factor (SRF) to stabilize the MRTF/SRF/CArG-box complex, leading to the expression of smooth muscle cell (SMC) genes such as α-smooth muscle actin (α-SMA) and collagens. These are the marker genes for myofibroblasts. Here, we show that the adhesion of cultured human skin fibroblasts (HSFs) to collagen reduces the myofibroblastic features. HSF adhesion to collagen suppresses the expression of CRP2 and CSRP2-binding protein (CSRP2BP [CRP2BP]) and reduces the expression of SMC genes. Although CRP2BP is known as an epigenetic factor, we find that CRP2BP also acts as an adaptor protein to enhance the function of CRP2 mentioned above. This CRP2BP function does not depend on its histone acetyltransferase activity. We also addressed the molecular mechanism of the reduced myofibroblastic features of HSFs on collagen. HSF adhesion to collagen inhibits the p38MAPK-mediated pathway, and reducing the p38MAPK activity decreases the expression of CRP2 and SMC genes. Thus, the activation of p38MAPK is critical for the myofibroblastic features. We also show evidence that CRP2 plays a role in the myofibroblastic transition of retinal pigment epithelial cells (RPEs). Like HSFs, such phenotypic modulation of RPEs depends on the p38MAPK pathway.Key words: CRP2, p38MAPK, MRTF, myofibroblasts, retinal pigment epithelial cells.

Keywords: CRP2; MRTF; myofibroblasts; p38MAPK; retinal pigment epithelial cells.

Fig. 1

Decreased myofibroblastic phenotype of HSFs by attachment to type I collagen (COLI) (A) HSFs were cultured on non-coated dishes (none), COLI thin film (COLI-film), or COLI-gel for 4 days. Whole-cell lysates were subjected to IB (left panel). The right graph shows the relative expression levels of each protein, MRTF-A, MRTF-B, SRF, and α-SMA. Their levels in HSFs cultured on non-coated dishes (none) were set at 100% (means ± SEMs of the results from multiple independent experiments, n = 3). Only the α-SMA expression significantly reduces in HSFs cultured on COLI-gel or COLI-film. ANOVA shows a significant difference in the expression levels of α-SMA among the three culture conditions (P<0.0001). Asterisks indicate P-values for multiple comparisons of the expression of α-SMA protein (pair to HSFs on non-coated dishes and HSFs on COLI-gel or COLI-film). (B) The expression of myofibroblast markers and the related factors. HSFs were cultured under the indicated conditions: non-coated dish (none), COLI-gel (gel), and COLI-film (film). RT-qPCR quantified the expression levels of the indicated mRNAs. Their levels in HSFs cultured on non-coated dishes were set at 100% (means ± SEMs of the results from multiple independent experiments, n = 3). ANOVA shows no significant difference in the expression levels of TGF-β1, TGF-β3, thymosin β4, and TGF-βR1-3 among the three culture conditions (P>0.05). Asterisks indicate the statistical significance of multiple comparisons between the indicated pairs (ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001: ****: P<0.0001).

Fig. 2

Effect of CRP2BP on the recruitment of MRTF-A/SRF/CRP2 to the CArG-box (A and B) Protein-protein interaction analysis using in vitro translated proteins. Mixtures of the indicated proteins (numbering proteins in the input panels) were subjected to IP/IB analysis as described in Materials and Methods. Interactions among MRTF-A, SRF, and CRP2 or CRP2BP, or these four proteins (A). The relative binding affinity of SRF to MRTF-A (right upper graph, A) and that of CRP2 to MRTF-A (right lower graph, A). The affinity levels in the IP2 and IP3 Mixes were normalized using the affinity level in the IP1 Mix as 100% (means ± SEMs of the results from multiple independent experiments, n = 3). ANOVA shows a significant difference in the binding affinity of SRF to MRTF-A among the three Mixes (IP1-IP3) (P<0.0001). Effect of CRP2BP on the interaction between SRF and CRP2 (B). The relative binding affinity of SRF to CRP2 (right graph, B). The affinity level in the IP2 Mix was normalized as described above. Quantified results are means ± SEMs of the results from multiple independent experiments (n = 3). (C) DNA affinity binding assay using in vitro translated proteins. Mixtures of the marked proteins (Mix a and Mix b) were pulled down with 3xCArG-box-Dynabeads (a1, a2, b1, b2) or control Dynabeads (cntl-beads) (a3, b3) in the absence or presence of free 3xCArG-box probes. IB shows the 3xCArG-box-bound proteins (left panel). The graphs show the quantification of the binding affinity of each protein to the 3xCArG-box-Dynabeads. In each experiment, the binding affinity in the absence of CRP2BP (a1) was set at 100% (means ± SEMs of the results from multiple independent experiments, n = 3). Asterisks indicate the statistical significance of multiple comparisons between the indicated pairs (ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001).

Fig. 3

Effect of CRP2BP on the transcriptional activity mediated by the CArG-box (A) Protein-protein interaction analysis using in vitro translated proteins. Mixtures of the indicated proteins (numbering proteins in the input panels) were subjected to IP/IB analysis as described in Materials and Methods. Interactions among MRTF-A, SRF, CRP2 and CRP2BP or CRP2BP637 were examined as described in the legend of Fig. 2A, B. The graph shows the relative binding affinity of SRF or CRP2 to MRTF-A. In each experiment, the respective affinity levels in the IP2 and IP3 Mixes were normalized using the affinity level in the IP1 Mix as 100% (means ± SEMs of the results from multiple independent experiments, n = 3). ANOVA shows a significant difference in the affinity levels of SRF and CRP2 to MRTF-A among the three binding conditions (P = 0.0122 for SRF and P = 0.0214 for CRP2). (B) Assessment of synergistic effects of MRTF-A, CRP2, and CRP2BP or CRP2BP637 on the CArG-box-dependent promoter activity in HSFs on non-coated dishes. Cells were transfected with 3xCArG-box-Luciferase reporter plasmid, pSVβ-gal, and the indicated plasmids or control plasmid. The promoter activity induced by exogenous MRTF-A alone was set at 100% (means ± SEMs of the results from multiple independent experiments, n = 3). ANOVA shows a significant difference in promoter activity among the ten different assays (P<0.0001). Asterisks indicate the statistical significance of multiple comparisons between the indicated pairs (ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001).

Fig. 4

Roles of the p38MAPK pathway for the myofibroblastic phenotype of HSFs (A) Suppression of p38MAPK activity by HSF attachment to COLI-film. IB analysis with whole-cell lysates from the indicated HSF cultured on non-coated dishes (none) or COLI-film (upper panel). The lower graph shows the phosphorylation ratios of p38MAPK (P-p38MAPK/p38MAPK) in the respective HSF cultures; the phosphorylation ratio in HSFs cultured on non-coated dishes was set at 100% (means ± SEMs of the results from multiple independent experiments (n = 3). (B) Effect of the p38MAPK inhibitor on the expression of SMC genes and CRPs 1 and 2. HSFs were cultured on non-coated dishes (none) or COLI-film in the presence of either vehicle (DMSO) (control [cntl]) or 10 μM SB20350 (SB) for 1 day. RT-qPCR quantified the expression levels of the indicated mRNAs. Their levels in HSFs treated with vehicles on non-coated dishes were set at 100% (means ± SEMs of the results from multiple independent experiments, n = 3). ANOVA shows a significant difference in the expression levels of α-SMA, COLI a1, and CRP2 (P<0.0001 for α-SMA and COLI a1, P = 0.0001 for CRP2) but not the expression levels of CRP1 (P = 0.9667) among the three culture conditions. Asterisks indicate the statistical significance of multiple comparisons between the indicated pairs (ns: P>0.05; *: P<0.05; **: P<0.01; ****: P<0.0001). (C) Schematic summary of the relationship between the p38MAPK pathway and SMC gene expression in HSFs cultured on non-coated dishes or COLI-film. HSF attachment to COLI-film decreases the activation of the p38MAPK pathway, which is necessary to activate the expression of CRP2 and its downstream SMC genes. + ~ +++++ indicate the relative activation levels of p38MAPK and the expression levels of α-SMA and COLI a1.

Fig. 5

Significance of CRP2 in TGF-β2-induced phenotype transition of hTERT-RPE1 cells (A) Assessment of synergistic effects of MRTF-A, CRP2, and CRP2BP or CRP2BP637 on the CArG-box-dependent promoter activity in hTETR-RPE1 cells. Cells were transfected with 3xCArG-box-Luciferase reporter plasmid, pSVβ-gal, and the indicated plasmids or control plasmid. The promoter activity induced by exogenous MRTF-A alone was set at 100% (means ± SEMs of the results from multiple independent experiments, n = 3). ANOVA shows a significant difference in the promoter activity among the ten different assays (P<0.0001). (B) Growing hTERT-RPE1 cells (70~80% confluent sate) were treated with either vehicle (PBS) containing 0.3% BSA (TGF-β2–) or TGF-β2 (5 ng/ml) for 1 day as described in Materials and Methods. RT-qPCR quantified the expression levels of the mRNAs for myofibroblast markers and related factors. Their levels in non-stimulated cells (TGF-β2–) were set at 100% (means ± SEMs of the results from multiple independent experiments, n = 3). (C) IB analysis confirms the up-regulation of the proteins whose mRNAs increase by TGF-β2 stimulation. Whole-cell lysates from hTERT-RPE1 cells cultured under the indicated conditions were subjected to IB. α-tubulin and GAPDH were used as loading controls. These are representative images from several examinations. (D) Activation of p38MAPK in TGF-β2-stimulated hTERT-RPE1 cells. IB analysis with whole-cell lysates from the indicated hTERT-RPE1 cells (TGF-β2– and TGF-β2+). The graph shows the phosphorylation ratios of p38MAPK (P-p38MAPK/p38MAPK) in the respective cells; the phosphorylation ratio in the control cells (TGF-β2–) was set at 100%. Each value represents the means ± SEMs of the results from multiple independent experiments (n = 3). (E) Effect of the p38MAPK inhibitor on the expression of α-SMA and CRP2. hTERT-RPE1 cells cultured in the presence of either vehicle (DMSO) [SB–] or 10 μM SB20350 (SB) [SB+] for 1 hour were stimulated with vehicle or TGF-β2 for 1 day as described above. Whole-cell lysates from the respective cells were subjected to IB with the indicated antibodies. The graph shows the quantification of the expression of α-SMA and CRP2 proteins. The expression levels of both proteins in the control cells (SB– and TGF-β2–) were set at 100% (means ± SEMs of the results from multiple independent experiments, n = 3). ANOVA shows a significant difference in the expression among the different cultures (P<0.0001 for α-SMA and P = 0.0014 for CRP2). Asterisks indicate the statistical significance of multiple comparisons between the indicated pairs (ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001).

Fig. 6

Significance of CRP2 in the cell motility and SMC gene expression in TGF-β2-stimulated hTERT-RPE1 cells hTERT-RPE1 cells were transfected with each siRNAs. One day after siRNA transfection, cells were stimulated with TGF-β2, as described in Materials and Methods. (A and B) Effect of KD of CRP2 on the motility of TGF-β2-stimulated hTERT-RPE1 cells. The cell motility was analyzed by wound healing assay as described in Materials and Methods. Representative images show cells immediately after scratching (0 hours) and at 16 hours (A). The motility of each cell was quantified (means ± SEMs of the results from multiple independent experiments, n = 3) (B). ANOVA shows a significant difference in the cell motility among three different culture conditions (P<0.0001). (C) RT-qPCR quantified the expression levels of the indicated mRNAs. The expression levels in control cells (cntl siRNA transfected cells without TGF-β2) were set at 100% (means ± SEMs of the results from multiple independent experiments, n = 3). ANOVA shows a significant difference in the expression levels of CRP2, α-SMA, and COLI a1 (P<0.0001 for CRP2, P = 0.0010 for α-SMA, P = 0.0002 for COLI a1). Asterisks indicate the statistical significance of multiple comparisons between the indicated pairs (ns: P>0.05; **: P<0.01; ****: P<0.0001).

Fig. 7

Conclusion remarks Activation of the p38MAPK pathway is critically important for the phenotypic transition from HSFs or RPEs to myofibroblasts. CRP2, whose expression depends on the activation of p38MAPK, activates the transcription mediated by MRTF/SRF/CArG-box followed by the induction of SMC gene expression, leading to the above-mentioned phenotypic transition. In this case, CRP2 acts as an adapter protein to stabilize the complex formed by SRF, MRTF, and CArG-box. Although CRP2BP is known as an epigenetic factor, CRP2BP also acts as an adaptor protein to promote the function of CRP2 mentioned above. Furthermore, cell adhesion to ECM suppresses the phenotypic transition to myofibroblasts because cell detachment from ECM induces the activation of p38MAPK.