Beta-catenin stabilization of skin fibroblasts causes fibrotic lesions by preventing adipocyte differentiation of the reticular dermis. Mus musculus

The Wnt/alpha-catenin pathway plays a central role in epidermal homeostasis and regeneration but how it affects fibroblast fate decisions is unknown. Here, we investigated the effect of targeted alpha-catenin stabilization in dermal fibroblasts. Comparative gene expression profiling of Sca1- and Sca1+ neonatal fibroblasts, from upper and lower dermis respectively, confirmed that Sca1+ cells had a pre-adipocyte signature and revealed differential expression of Wnt/alpha‐catenin-associated genes. By targeting all fibroblasts or selectively targeting Dlk1+ lower dermal fibroblasts, we found that -catenin stabilization between E16.5 and P2 resulted in a reduction in the dermal adipocyte layer with a corresponding increase in dermal fibrosis and an altered hair cycle. The fibrotic phenotype correlated with a reduction in the potential of Sca1+ fibroblasts to undergo adipogenic differentiation ex vivo. Our findings indicate that Wnt/alpha-catenin signaling controls adipogenic cell fate within the lower dermis, which potentially contributes to the pathogenesis of fibrotic skin diseases. Overall design: The dermis was separated from back skin of PDGFRAeGFP postnatal pups (P2) by incubation with thermolysin (0.25 mg/ml) (Sigma T7902) overnight at 4° and further processed as previously described (Collins et al., 2011). Cells were labeled in PBS + 10% FBS TruStain fcX anti-mouse blocking buffer with the following antibodies: anti-mouse Ly-6A/E (Sca-1)-Alexa Fluor-700 17. Two populations of cells were collected in triplicate. The PDGFRaH2BeGFP/Sca1- and the PDGFRaH2BeGFP/Sca1+. RNA was isolated and prepared for microarray analysis and hybridized to Affymetrix MG430.2A arrays. C, digested in DMEM + 10% FBS containing 2.5 mg/mL collagenase I (Gibco 17100- 017), and further processed

Wnt/α-连环蛋白通路（Wnt/alpha-catenin pathway）在表皮稳态与再生中发挥核心作用，但其如何调控成纤维细胞的命运决定尚未明确。本研究探究了靶向稳定真皮成纤维细胞中α-连环蛋白的效应。对分别源自真皮上层与下层的Sca1-和Sca1+新生鼠成纤维细胞进行比较基因表达谱分析，证实Sca1+细胞具有前脂肪细胞特征，并揭示了Wnt/α-连环蛋白相关基因的差异表达。通过靶向所有成纤维细胞，或选择性靶向Dlk1+下层真皮成纤维细胞，我们发现，在胚胎第16.5天（E16.5）至出生后第2天（P2）期间稳定β-连环蛋白，可导致真皮脂肪层减少，同时伴随真皮纤维化程度升高与毛发周期改变。该纤维化表型与体外实验中Sca1+成纤维细胞的成脂分化潜能降低相关。本研究结果表明，Wnt/α-连环蛋白信号通路可调控下层真皮内的成脂细胞命运，这一过程或参与纤维化性皮肤病的发病机制。整体实验设计：取PDGFRAeGFP阳性新生仔鼠（P2）的背部皮肤，分离真皮层：将组织置于0.25mg/ml嗜热菌蛋白酶（Sigma T7902）中4℃孵育过夜，随后按照此前报道的方法（Collins等，2011）进行后续处理。将细胞置于含10%胎牛血清（FBS）的PBS缓冲液中，使用TruStain fcX抗小鼠封闭缓冲液搭配以下抗体进行标记：抗小鼠Ly-6A/E（Sca-1）-Alexa Fluor-700 17。分两个细胞群进行三次生物学重复收集：PDGFRaH2BeGFP/Sca1-细胞群与PDGFRaH2BeGFP/Sca1+细胞群。提取细胞总RNA并制备用于微阵列分析，将样本杂交至Affymetrix MG430.2A芯片。C组：使用含2.5mg/mlⅠ型胶原酶（Gibco 17100-017）的DMEM培养基（含10%FBS）进行消化，随后进行后续处理。