Lineage tracing mediated by Cre-recombinase activity

A

Deletion of the Major Bullous Pemphigoid Epitope Region of Collagen XVII Induces Blistering, Autoimmunization, and Itching in Mice

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering skin disease with a characteristic of pruritus and blistering. BP patients carry inflammation-triggering autoantibodies against the collagen XVII (ColXVII, also known as BP180) juxtamembraneous extracellular noncollagenous 16A (NC16A) domain involved in ectodomain shedding. Deletion of the corresponding NC14A region in a genetically modified mouse model (ΔNC14A) decreased the amount of ColXVII in skin, but it did not prevent ectodomain shedding. Newborn ΔNC14A mice had no macroscopic phenotypic changes. However, subepidermal microblisters, rudimentary hemidesmosomes, and loose basement membrane zone were observed by microscopy. ΔNC14A mice grow normally, but at around 3 months of age they start to scratch themselves and develop crusted erosions. Furthermore, perilesional eosinophilic infiltrations in the skin, eosinophilia, and elevated serum IgE levels are detected. Despite the removal of the major BP epitope region, ΔNC14A mice developed IgG and IgA autoantibodies with subepidermal reactivity, indicating autoimmunization against a dermo-epidermal junction component. Moreover, IgG autoantibodies recognized a 180-kDa keratinocyte protein, which was sensitive to collagenase digestion. We show here that ΔNC14A mice provide a highly reproducible BP-related mouse model with spontaneous breakage of self-tolerance and development of autoantibodies

Transmembrane Collagen XVII Modulates Integrin Dependent Keratinocyte Migration via PI3K/Rac1 Signaling

<div><p>The hemidesmosomal transmembrane component collagen XVII (ColXVII) plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits β4 and β1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K) activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the β4 integrin subunit and the focal adhesion kinase (FAK) as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma.</p></div

Enhanced spreading and actin dynamics in <i>Col17a1^−/−</i> keratinocytes.

<p><b>A</b>, Keratinocytes derived from wild type (Ctrl) and <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} mice were grown on LN332 coated chamber slides for 30 minutes, fixed and processed for indirect immunofluorescence staining with an actin antibody. The insert represents one enlarged <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} cell. Arrowheads indicate the presence of multiple lamellipodia visible by tightly dense actin staining. <i>Scale bar  = 10 µm</i>. <b>B</b>, The graph shows the cell size in µm². The size of <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes was about 40% larger than that of Ctrl cells (<i>n = 30</i>; cells of two individuals per genotype have been analyzed). Data are shown as mean ± SEM; ***<i>p</i><0.001. <b>C</b>, Semi-confluent Ctrl and <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes were fixed with 4% PFA and incubated with rhodamin-conjugated phalloidin for 1 hour. Phalloidin-staining indicated a higher number of stress fibers and reduced cortical actin in <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes.</p

Upregulation of α6β4 and β1 integrins in <i>Col17a1^−/−</i> skin.

<p><b>A</b> and <b>B</b>, Skin lysates from WT and <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} mice were immunoblotted with indicated antibodies. The graphs combine the quantification for β4 protein expression of four individuals per genotype (<b>A</b>) and β1 protein expression of three individuals (<b>B</b>). *<i>p</i><0.05. <b>C</b>, Quantitative RT-PCR of primary wild type and <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes (cells of four individuals per genotype have been analyzed; number of independent measurements  = 3). Data are shown as mean ± SEM; *<i>p</i><0.05.</p

Altered cell detachment in the absence of ColXVII.

<p><b>A</b>, Trypsin-based cell detachment assay. Confluent cell layers of keratinocytes derived from wild type (Ctrl) and <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} mice were treated with trypsin/EDTA (0.05%/0.02%) for indicated time points (cells of three individuals per genotype have been analyzed; number of independent measurements  = 5). <b>B</b>, For the centrifugal force-based cell detachment assay cells were allowed to adhere on laminin 332 (LN332) for 10 minutes before measuring the strength of the adhesion at indicated centrifugal forces (cells of three individuals per genotype have been analyzed; number of independent measurements  = 3). For both assays adherent cells were stained with 0.5% crystal-violet, lysed with 1% SDS, and the percentage of adherent cells was determined spectrophotometrically at 540 nm. Data are shown as mean ± SEM; **<i>p</i><0.01.</p

β4 integrin subunit is functionally involved in cell adhesion and spreading of <i>Col17a1^−/−</i> cells.

<p><i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes were transduced with empty pLKO vector (mock) and two different shRNA to β4 integrin subunit (β4kd#1 and β4kd#2). <b>A</b>, The cells were lysed and equal amounts of total protein were immunoblotted with indicated antibodies. <b>B</b>, Confluent layers of <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} cells were subjected to trypsin/EDTA detachment assay (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087263#pone-0087263-g001" target="_blank">Figure 1</a>). The percentage of adherent cells are shown as mean ± SEM (number of independent measurements  = 3); <i>*p</i><0.05. <b>C</b>, Cells were grown on LN332 coated chamber slides for 30 minutes, fixed and processed for indirect immunofluorescence with an actin antibody. The graph shows the cell area in µm² (<i>n = </i>35). The data are shown as mean ± SEM. *<i>p</i><0.05. <b>D</b>, <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes were treated with DMSO (Ctrl) or different phospho-FAK inhibitors (PF 573228 [5 µM] and Inhibitor 14 [1 µM]) for 6 hours and thereafter subjected to trypsin/EDTA detachment assay. The data are shown as mean ± SEM (cells of three individuals have been analyzed; number of independent measurements  = 5).</p

A schematic model of the migratory phenotype of <i>Col17a1^−/−</i> keratinocytes.

<p>In wild type cells the endodomain of ColXVII binds to the intracellular domain of the β4 integrin subunit. In <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes, the genetic ablation of ColXVII leads to increased expression and phosphorylation (S1356) of the β4 subunit and to phosphorylation of FAK (Y397), which, in turn, enhance PI3K activity and induce undirected cell migration via Rac1 activation.</p