Protein phosphatase and tensin homolog (PTEN) expression is reduced in dermal fibroblasts isolated from patients with diffuse cutaneous systemic sclerosis, a fibrotic autoimmune disease. In support of this finding, deletion of the PTEN gene in the dermal fibroblasts of mice has been shown to result in skin fibrosis and in vivo overexpression of connective tissue growth factor (CTGF; CCN2), a proadhesive matricellular protein; however, whether CCN2 is required for the fibrosis caused by loss of PTEN is unclear. This study was undertaken to investigate the role of CCN2 in fibrosis caused by reduced PTEN expression.
We generated conditional knockout mice in which PTEN was deleted in fibroblasts, either alone or in combination with CCN2. Skin samples were collected for histologic examination, immunohistochemical analysis, and collagen assay.
Loss of CCN2 resulted in resistance to the increases in collagen production and myofibroblast recruitment that are caused by loss of PTEN. CCN2 deficiency did not impair Akt phosphorylation or the increases in the intensity of proliferating cell nuclear antigen staining that were caused by loss of PTEN.
These data are consistent with the notion that CCN2 is required for particular aspects of the fibroproliferative response; therapeutic strategies blocking CCN2 may be of clinical benefit in combating fibrotic disease.
Tissue fibrosis is associated with several diseases, including scleroderma (systemic sclerosis [SSc]) (). Fibrosis is mediated by the abnormal persistence of myofibroblasts, a specialized form of fibroblast characterized by the overexpression of α-smooth muscle actin (α-SMA), a highly contractile protein (). Myofibroblasts are characterized by elevated adhesion to and contraction of extracellular matrix and adhesive signaling of kinases, including focal adhesion kinase (FAK)/phosphatidylinositol 3-kinase (PI3K)/Akt (). Expression of the phosphatase and tensin homolog (PTEN) protein, a dual protein/lipid phosphatase that suppresses the activation of PI3K/Akt signaling, is reduced in SSc fibroblasts (). Genetic deletion of PTEN in skin fibroblasts has been found to result in progressive dermal fibrosis in vivo ().
Connective tissue growth factor (CTGF; CCN2), a member of the CCN family of matricellular proteins (), is overexpressed in PTEN-deficient mice and fibroblasts (). CCN2–conditional-knockout mice were recently used to show that CCN2 was required for fibrosis in the bleomycin model of skin SSc (). However, bleomycin is an imperfect model of SSc (), and it is necessary to study additional models of fibrosis in SSc to validate CCN2 as a potential antifibrotic target. In particular, it is unknown whether CCN2 is necessary for the fibrosis caused by loss of PTEN expression in fibroblasts. In this study, we assessed whether CCN2 is required for the skin fibrosis caused by PTEN deficiency. Our results reveal useful insights into the molecular mechanisms underlying fibrosis and suggest that CCN2 may only modulate particular aspects of the fibrotic response.
MATERIALS AND METHODS
Mice in which PTEN was deleted in fibroblasts and mice in which both PTEN and CCN2 were deleted in fibroblasts were generated essentially as previously described ([4, 6]). Briefly, loxP-Pten mice were obtained from The Jackson Laboratory. LoxP-Ccn2 mice and mice expressing tamoxifen-dependent Cre recombinase under the control of the fibroblast-specific regulatory sequence from the proα2(I) collagen gene were generated as previously described ([6, 8]). Mice hemizygous for Cre and homozygous for loxP-Pten were administered tamoxifen (1 mg each for 5 days) or corn oil at 21–24 days of age to generate mice in which PTEN was specifically deleted in fibroblasts or control mice, respectively. Mice hemizygous for Cre and homozygous for loxP-Pten and loxP-Ccn2 were similarly used to generate mice in which PTEN and CCN2 were deleted. Deletion of PTEN and/or CCN2 was tested by polymerase chain reaction genotyping as described previously ([4, 6]). Mice were killed by CO2 inhalation at the various time points. Forty-five days after the last tamoxifen injection (), skin samples were collected for histologic examination, immunohistochemical analysis, and collagen assays. This time point has previously been shown to be optimal for development of the PTEN-deficient phenotype (). All animal experiments received ethical approval from the University of Western Ontario's Animal Care Committee.
Immunohistochemical analysis and assessment
Skin tissue sections (0.5 μm) were cut using a microtome (Leica) and collected on Superfrost Plus slides (Fisher Scientific). Sections were dewaxed in xylene and rehydrated by successive immersion in descending concentrations of alcohol. When indicated, tissue was stained with Harris' hematoxylin and eosin (H&E) or trichrome. Skin thickness was measured using Northern Eclipse image analysis software (Empix Imaging).
Sections were also subjected to immunofluorescence staining. Tissue sections were blocked by incubation with 5% bovine serum albumin/0.1% Triton X-100 in phosphate buffered saline (PBS) for 1 hour and then incubated with primary antibodies under humidified conditions at 4°C overnight. The primary antibodies used in these studies were as follows: anti–α-SMA (1:1,000 dilution; Sigma), anti-CCN2 (1:200; Santa Cruz Biotechnology), anti–proliferating cell nuclear antigen (anti-PCNA) (1:500 dilution; Abcam), and anti-pAkt (1:100 dilution; Cell Signaling). After primary antibody incubation, sections were washed with PBS and incubated with appropriate fluorescent secondary antibodies (Jackson ImmunoResearch) for 1 hour at 37°C. Sections were washed with PBS, mounted using DAPI, and photographed using a Zeiss fluorescence microscope and Northern Eclipse software. The percentage of positive cell staining was calculated by the number of cells/mm2 using Northern Eclipse software. To quantify collagen, hydroxyproline was detected in tissue hydrolysates using a Total Collagen Kit, according to the instructions of the manufacturer (QuickZyme Biosciences).
Western blot analysis
Mouse fibroblasts were cultured by explant in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Life Technologies) and subjected to Western blot analysis as previously described (). The antibodies used in these studies were as follows: anti–α-SMA (1:5,000 dilution), anti-CCN2 (1:500 dilution), anti-PTEN (1:500 dilution; Abcam), anti–β-actin (1:5,000 dilution; Sigma), and anti-Akt (1:1,000 dilution) and anti-pAkt (1:1,000 dilution) (both from Cell Signaling). Protein bands were detected by chemiluminescence (Fisher).
Role of CCN2 in dermal fibrosis resulting from loss of PTEN expression in skin fibroblasts
Previously, we demonstrated that spontaneous skin fibrosis, including elevated CCN2 expression, occurred in mice with fibroblasts deficient in PTEN expression (). To assess whether loss of CCN2 expression in fibroblasts was required for this phenomenon, mice in which PTEN was deleted in fibroblasts (Ptenfl/fl) or wild-type mice without PTEN deletion were generated as described previously () and above. Moreover, to assess whether CCN2 was required for the fibrosis caused by loss of PTEN, mice in which both PTEN and CCN2 were deleted in fibroblasts (Ptenfl/fl;Ccn2fl/fl) were similarly generated. Consistent with prior data (), compared to their wild-type counterparts, PTEN-deficient mice showed increased dermal thickness as revealed by H&E staining (Figure 1A), elevated dermal collagen production as visualized by trichrome staining and colorimetric enzyme-linked immunosorbent assay (Figure 1B), and myofibroblast formation as measured by indirect immunofluorescence analysis with an anti–α-SMA antibody (Figure 2). Examination of mice in which both PTEN and CCN2 were deleted revealed that loss of CCN2 at least partially prevented the elevated skin thickness, collagen production, and myofibroblast formation seen in PTEN-deficient animals (Figures 1A and B and Figure 2). Collectively, these data suggest that, in fibroblasts, CCN2 contributes to the fibrosis caused by loss of PTEN expression.
Lack of CCN2 involvement in the elevated Akt phosphorylation and increased PCNA staining observed in PTEN-deficient mice
Akt is a target of PTEN (), and previous data have indicated that the overexpression of CCN2 in PTEN-deficient mouse fibroblasts was mediated by Akt (). To further elucidate which aspects of the fibrotic phenotype in PTEN-deficient mice are mediated by CCN2, we assessed the phosphorylation status of Akt. The significant increase in the number of dermal fibroblasts expressing pAkt in Ptenfl/fl mice compared to controls was not affected by loss of CCN2 (Figure 3A). This indicates that CCN2 was not responsible for elevated Akt phosphorylation in PTEN-deficient mice and is consistent with the notion that CCN2 overexpression in PTEN-deficient fibroblasts is downstream of Akt (). Moreover, the elevated intensity of PCNA staining observed in PTEN-deficient mice () was not reversed by loss of CCN2 (Figure 3B), suggesting that CCN2 was also not involved in the proliferative phenotype in Ptenfl/fl mice. Western blot analysis of dermal fibroblasts isolated from wild-type controls, Ptenfl/fl mice, and Ptenfl/fl;Ccn2fl/fl mice confirmed that CCN2 was not responsible for the elevated Akt phosphorylation observed in Ptenfl/fl mice (Figure 3C).
PTEN deficiency is a hallmark of SSc fibroblasts ([4, 10]), and loss of PTEN expression in fibroblasts has been shown to be sufficient for fibrogenesis in vivo (). Moreover, reintroduction of PTEN into fibroblasts cultured from the dermal lesions of patients with diffuse cutaneous SSc reversed the fibrotic phenotype, as visualized by type I collagen and CCN2 expression (). These results indicated that PTEN normally suppresses fibrogenic responses in vivo, and loss of PTEN is sufficient for fibrogenesis. Therefore, we hypothesized that PTEN-knockout mice could represent a new model for the fibrosis observed in SSc.
PI3K/Akt is a target of PTEN (), and the overexpression of CCN2 in PTEN-deficient mouse fibroblasts is dependent on PI3K/Akt (). In this study, we extended these data to test the hypothesis that CCN2 was responsible for the fibroproliferative phenotype observed in PTEN-deficient mice. We also showed that CCN2 expression in fibroblasts was responsible for elevated dermal thickness and increased collagen production in the skin and lungs of mice in which PTEN has been deleted in fibroblasts. Moreover, we demonstrated that, whereas CCN2 expression by fibroblasts contributed to the presence of myofibroblasts in the dermis of mice in which PTEN was deleted in fibroblasts, CCN2 deletion did not affect the elevated Akt phosphorylation and increased intensity of PCNA staining in PTEN-deficient mice. These data are consistent with the notion that alterations in PTEN expression contribute to lung fibrosis in vivo (). Results of our study also support previous observations that CCN2 is important for bleomycin-induced skin and lung fibrosis ([4, 12]) and the hypothesis that CCN2 plays an important, central role in fibrogenesis ([13-15]). The fibrosis caused by loss of PTEN could be caused by increased survival of myofibroblasts or decreased apoptosis, although differences in apoptosis are not apparent in the skin of PTEN-deficient mice as compared to the skin of wild-type mice (Liu S, Leask A: unpublished observations).
Although induced by transforming growth factor β (TGFβ), CCN2 does not appear to be a downstream mediator of TGFβ (); rather, CCN2 may act either as a cofactor enhancing the fibrogenic ability of TGFβ or by recruiting pericyte-like progenitor cells to the fibrotic lesion ([4, 13]). Further efforts are necessary to ascertain the precise role of CCN2 in fibrogenesis.
Our results suggest that the fibroproliferative aspect of fibrosis can be separated mechanistically from the myofibroblast/collagen overproduction aspect of fibrosis. CCN2 may therefore represent a common downstream mediator of at least certain features of fibrosis and may be a suitable target for antifibrotic drug intervention.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Leask had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Liu, Parapuram, Leask.