Critical <i>in vivo</i> roles of WNT10A in wound healing by regulating collagen expression/synthesis in <i>WNT10A</i>-deficient mice

<div><p>Background</p><p>We have reported that WNT10A plays a critical role in the growth of fibroblasts/myofibroblasts and microvascular endothelial cells, i.e.; wound healing/scarring. To ascertain the <i>in vivo</i> regulatory, central functions of WNT10A, we examined the net effects of WNT10A depletion using <i>WNT10A</i>-deficient mice (<i>WNT10A</i>^–/–).</p><p>Methods and results</p><p>We generated <i>WNT10A</i>^–/–mice, displaying a range of unique phenotypes of morpho/organogenetic failure, such as growth retardation, alopecia, kyphosis and infertility, and then focused on the functions of WNT10A in wound healing. We subjected C57BL/6J wild-type (WT) or <i>WNT10A</i>^–/–mice to skin ulcer formation. The <i>WNT10A</i>^–/–mice had significantly larger injured areas and delayed wound healing, which were associated with (a) a smaller number of fibroblasts/myofibroblasts and microvessels; and (b) more reduced expression and synthesis of collagen, compared with WT mice with intact WNT10A expression, especially in those with activated myofibroblasts.</p><p>Conclusions</p><p>These observations indicate that WNT10A signaling can play a pivotal in vivo role in wound healing by regulating the expression and synthesis of collagen, as one of fibrogenic factors, at least in part, and critical <i>in vivo</i> roles of WNT10A-mediated effective wound healing are extremely closely associated with collagen expression.</p></div

Role of WNT10A-Expressing Kidney Fibroblasts in Acute Interstitial Nephritis

<div><p>WNT signaling mediates various physiological and pathological processes. We previously showed that WNT10A is a novel angio/stromagenic factor involved in such processes as tumor growth, wound healing and tissue fibrosis. In this study, we investigated the role of WNT10A in promoting the fibrosis that is central to the pathology of acute interstitial nephritis (AIN). We initially asked whether there is an association between kidney function (estimated glomerular filtration rate; eGFR) and WNT10A expression using kidney biopsies from 20 patients with AIN. Interestingly, patients with WNT10A expression had significantly lower eGFR than WNT10A-negative patients. However, changes in kidney function were not related to the level of expression of other WNT family members. Furthermore, there was positive correlation between WNT10A and α-SMA expression. We next investigated the involvement of WNT10A in kidney fibrosis processes using COS1 cells, a kidney fibroblast cell line. WNT10A overexpression increased the level of expression of fibronectin and peroxiredoxin 5. Furthermore, WNT10A overexpression renders cells resistant to apoptosis induced by hydrogen peroxide and high glucose. Collectively, WNT10A may induce kidney fibrosis and associate with kidney dysfunction in AIN.</p></div

<i>WNT10A</i>^–/–KO skin wound showing reduced fibrosis/fibrogenesis associated with the decreased expression of stromagenesis-related genes.

<p><b>A)</b> Representative pictures of Masson’s trichrome staining (Scale bars = 100 μm) show that collagen deposits (<i>blue</i>-stained) were markedly smaller with more reduced fibrosis in the injured incurable skin lesions of KO mice than in those of WT mice (n = 8 mice per group). In addition, an immunofluorescence study (Scale bars = 20 μm) reveals that a large number of WT spindle (myo)fibroblasts (<i>blue-stained</i> in nuclei) have significant expression of Type I/III collagen at day 10 post-skin injury, which is very rarely or not observed in KO (myo)fibroblasts. <b>B)</b> On a quantitative analysis, the <i>blue</i>-stained collagen content of KO injured skin was found to be markedly lower than in WT skin. <b>C)</b> Correspondingly, real-time RT-PCR showed that the mRNA expression of <i>Type I</i> and <i>Type III collagen</i> was significantly lower in the KO skin of the day-3 wound healing model than in the WT skin. Values are means ± SE and were normalized for 18s rRNA expression (real-time RT-PCR). *<i>P</i> < 0.05, ***<i>P <</i> 0.0001.</p

eGFR according to WNT proteins.

<p>eGFR; median [interquartile range] mL/min per 1.73 m² above (on biopsy), below (on discharge).</p

WNT10A overexpression enhances fibronectin expression in COS1 cells.

<p>(A) The expression of ECM components in WNT10A-overexpressing cells. Western analysis was performed using whole lysates obtained from control and WNT10A-overexpressing cells (COS1-10A). (B) Relative expression of fibronectin, type 1 collagen, and type 3 collagen, which were normalized to β-actin expression. The black bar shows control cells, white and gray bars showed COS1-10A-cl.1 and COS1-10A-cl.2 respectively. (C) Immunofluorescence staining for WNT10A (green), fibronectin (FN) (green), and DAPI (blue). Control cells treated with normal medium (left panel), control cells treated with supernatant (sup.) of WNT10A-overexpressing cells (middle panel), and COS1-10A cells treated with normal medium (right panel). Each cell was cultured for 72 hours. All photos were taken at 200×. Scale bar is 100 µm. (D) Immunohistochemical staining for fibronectin (brown) in AIN tissues. Kidney tissues from WNT10A-negative (left panel) or -positive (right panel) patients are shown. Arrows indicate fibronectin-positive cells. All photos were taken at 200×. Scale bar is 100 µm.</p

Our generated <i>WNT10A</i>^–/–mice (KO) showing a range of unique phenotypes of morpho/organogenetic failure.

<p><b>A)</b> Under basal conditions, morphological examinations between the 12-week-old wild-type (WT) and KO mice (n = 10 mice per group) show that the KO mice are grossly smaller in size and have marked alopecia and kyphosis compared to WT. <b>B)</b> Representative whole-body X-ray confirms significant growth retardation in the KO mice, accompanied by marked kyphosis and many bony hyperlucent areas (arrows), in contrast to WT mice (n = 10 mice per group). <b>C)</b> From 1 to 12 months of age (whole one year), both male and female KO display significantly smaller body weights, as reflected by more essential growth retardation, than WT (n = 10 mice per group). <b>D)</b> The bone length and bone mineral density (BMD) in both male and female 12-week-old KO mice are significantly lower than those of WT mice (n = 10 mice per group), corresponding to the gross and imaging findings. E) Female KO mice with a phenotype of infertility demonstrate that the tissue of the grossly smaller KO ovary contains markedly fewer (or no) follicles than the ovary of WT mice on representative H&E sections. F) The number of follicles is significantly smaller in the KO ovary than in the WT ovary (n = 10 mice per group). Scale bars = 100 μm. Values are means ± SE. *<i>P <</i> 0.05, **<i>P <</i> 0.001, ***<i>P <</i> 0.0001.</p

The deficiency of <i>WNT10A</i> suppressed cell proliferation activity in wound healing mice model.

<p>Immunofluorescence staining showed that a significantly larger number of histone H3-positive cells (<i>green-stained</i>) were observed in WT mice wound skin, compared to <i>WNT10A</i>^–/–mice (n = 3 per group). The fluorescence of histone H3 was located in nuclei (inset) (<i>blue-stained</i> in nuclei). Scale bars = 100 μm.</p

Patient characteristics.

<p>AIN; acute interstitial nephritis, MPA; microscopic polyangiitis, HD; hemodialysis.</p><p>S.D; standard deviation.</p

The relationship between WNT family gene expression and kidney function.

<p>(A) Patient selection method to evaluate eGFR among AIN patients. eGFR (mL/min per 1.73 m²) of AIN patients with (B) WNT1, (C) WNT3, (D) WNT4, and (E) WNT10A. (B) WNT1 negative-expression; n = 9, positive-expression; n = 11 (C) WNT3 negative-expression; n = 12, positive-expression; n = 8 (D) WNT4 negative-expression; n = 15, positive-expression; n = 5 (E) WNT10A negative expression; n = 10, positive-expression; n = 10. Light gray box indicates eGFR of patients without WNT protein expression, and dark gray box indicates eGFR of patients with WNT protein expression. The left panels show kidney function on kidney biopsy, and right panels show kidney function on discharge. Data of each category is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103240#pone-0103240-t002" target="_blank">Table 2</a>.</p

WNT10A protected fibroblasts against high glucose stress.

<p>(A) Proliferation assay. Control and WNT10A-overexpressing cells (COS1-10A-cl.1) (2×10⁴ cells/well) were seeded into a 12-well plate, and incubated in normal DMEM. After 24 hours, cells (0hr) were harvested, and other cells were cultured in each medium (5.5 mM, 11 mM, and 22 mM of glucose concentration) for 72 hours. Proliferative rates for 72 hours were compared. Black bar (5.5 mM), white bar (11 mM), gray bar (22 mM). All values are the means of at least three independent experiments. Bars indicate standard deviation. *p<0.001 vs 5.5 mM, **p<0.001 vs 5.5 mM and 11 mM, #p<0.05 vs 5.5 mM (B) Caspase and PARP expression. Whole lysates obtained from control and WNT10A-overexpressing cells treated with each medium (5.5 mM, 11 mM, and 22 mM of glucose concentration) were analyzed by western blotting.</p