Abstract
AbstractIndividuals with Junctional Epidermolysis Bullosa (JEB), a rare genetic skin disease characterised by loss of function mutations in the Laminin332 (Lam332), do not survive beyond their first birthday. Here we report that loss of Lam332 leads to absence of cholesterol lipid from the epidermisin vitroandin vivo. Stable knockdown of Lam332 chains (LAMA3, LAMB3 and LAMC2) was established using shRNA and were used to develop 3D skin equivalents. Changes in lipid synthesis were assessed by western blot analysis and immunohistochemistry. Findings were confirmed in an inducible mouse model of Lamα3 (Lama3flox/flox/K14CreERT) and in anonymized, archival human tissue: JEB skin and normal age-matched controls. Further lipid analysis was explored using lipidomics in 3D skin equivalents and mouse tissue. Cholesterol biosynthesis genes were increased with loss of Lam332in vitro, however a decrease in nile red lipid staining was observed in Lamα3 mouse (n = 6) and in JEB patient skin (n = 7). Further changes to the epidermal lipid profile with loss of Lam332 was confirmed with lipidomic analysis of Lamα3 mouse epidermis and Lam332 skin equivalents. Cholesterol transport within Lam332 KD keratinocytes was revealed to be disrupted, which in keratinocytes is dependent on the actomyosin network, which was reversed with recombinant human Lam332. In conclusion these findings suggest a role for Lam332 in lipid metabolism in the skin and a broader role in epidermal homeostasis and barrier formation. Restoration of cholesterol transport in JEB patients offers the potential to improve the skin barrier and survival.
Publisher
Cold Spring Harbor Laboratory