Lysyl hydroxylase 3 localizes to epidermal basement membrane and Is reduced in patients with Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention

<em>NOTCH1</em> mutations occur early during cutaneous squamous cell carcinogenesis

This work was funded by Cancer Research UK and The British Skin Foundation. IML is funded by the European Research Council and a strategic grant from the Wellcome Trust

Bone marrow transplantation increases type VII collagen but does not change LH3 at the basement membrane in RDEB patients.

<p><b>(a</b>) LH3 immunoreactivity (red) does not change after bone marrow transplantation in two RDEB patients. (<b>b</b>) Type VII collagen expression (green) is increased after bone marrow transplantation. d = day, yr = year. Graph shows morphometric quantification of LH3 and type VII collagen signal intensities, +/- standard deviation. * = p < 0.001, ** = p < 0.0001, *** = p < 0.00001, students-T test.</p

Endogenous but not exogenous type VII collagen increases LH3 expression in RDEB keratinocytes.

<p><b>(a</b>) Retroviral delivery of type VII collagen in RDEB14 keratinocytes increases LH3 expression by immunofluorescence. (<b>b</b>) Type VII collagen increases LH3 in RDEB14 keratinocytes in cell extracts and conditioned media by immunoblotting. (<b>c</b>) siRNA depletion of <i>COL7A1</i> reduces LH3 expression in normal keratinocytes compared with non-targeting control (NT). (<b>d</b>) RDEB14 keratinocytes grown in conditioned media collected from RDEB14 keratinocytes overexpressing type VII collagen do not have increased LH3 expression compared with conditioned media collected from RDEB14 keratinocyte empty vector controls. (<b>e</b>) RDEB14 keratinocytes grown on cell matrix derived from type VII collagen overexpressing RDEB14 keratinocytes do not show increased LH3 expression compared with empty vector control treated cells. Empty Vector or EV = empty vector cells, C7A1 = <i>COL7A1</i> over expressing cells.</p

Type VII collagen binds LH3 <i>in vitro</i> and <i>in vivo</i> at the basement membrane in normal skin.

<p><b>(a</b>) LH3 interacts with type VII collagen (C7) <i>in vitro</i>. Cell extracts from RDEB keratinocytes retrovirally expressing COL7A1 were immunoprecipitated (IP) with a C7 antibody (lane 1) and immunoblotted for LH3 co-precipitation. Controls include a non-specific IgG (lane 2), protein G beads alone (no antibody, lane 3) or C7 IP in COL7A1 null keratinocyte extracts (lane 4). Lanes 5 and 6 represent the inputs from COL7A1 expressing and null keratinocyte extracts respectively. (<b>b</b>) Proximity Ligation Assay demonstrates the close interaction of antibodies raised against LH3 and type VII collagen <i>in vivo</i> at the basement membrane in normal skin (top left panel, red signal) which is greatly reduced or absent in RDEB skin, with LH3 antibody only or with antibodies against LH3 and LN332 γ-2 chain. (<b>c</b>) Postembedding immunoelectron microscopy of human skin shows immunogold labeling beneath epidermal tissue using the LH7.2 mouse monoclonal antibody that recognizes the type VII collagen C-terminus (C7, upper panel, 5nm particles, arrowheads). LH3 polyclonal antisera (LH3, lower panel, 10nm particles, white arrows) localized beneath the epidermal tissue in an overlapping distribution close to anchoring fibrils (arrowheads, lower panel). Graph shows morphometric analysis of distance from plasma membrane (indicated by *) of immunogold particles. There were statistically significant differences (p < 0.01) observed between the depth of the two PLOD3 epitopes and both collagen VII epitopes. LH3/PLOD3 R = rabbit polyclonal antibody, M = mouse monoclonal antibody.</p

LH3 localizes to the basement membrane and is reduced in RDEB.

<p><b>(a)</b> LH3 immunohistochemistry reveals bright linear reactivity at the basement membrane in normal skin (top left panel) which is discontinuous and greatly reduced in RDEB skin. Graph shows morphometric quantification of LH3 signal intensity, +/- standard deviation. * = p < 0.01, ** = p < 0.0005, students-T test. (<b>b</b>) Type VII collagen immunohistochemistry shows bright linear reactivity at the basement membrane in <i>COL7A1</i> +/- newborn murine skin (+/- C7, top left panel) which is absent in <i>COL7A1</i> -/- newborn murine skin (bottom left panel). LH3 immunohistochemistry shows bright linear reactivity at the basement membrane in <i>COL7A1</i> +/- murine skin (top right panel) which is reduced in <i>COL7A1</i> -/- murine skin (bottom right panel). (<b>c</b>) LH3 protein is reduced in cell extracts and significantly reduced in conditioned media from RDEB keratinocytes (RDEB1K, RDEB14K and RDEB2K) compared with normal keratinocytes (NHK, K16 and K17). (<b>d</b>) Fibroblast cultures isolated from RDEB patients express significantly less LH3 compared with cultures of normal fibroblasts (NHF). Graphs show average densitometry of LH3 protein normalized to GAPDH (using ImageJ version 1.48, <a href="http://imagej.nih.gov/ij/" target="_blank">imagej.nih.gov/ij/</a>) for RDEB or normal controls. p values calculated using student-T test and error bars show +/- standard deviation.</p