Abstract
The skin provides a barrier function mediated by a combination of cornified epithelial cells, cell-cell adhesions and epidermal lipids. The barrier function is compromised in old age which is exemplified by features such as improper lipid metabolism and flattened dermal-epidermal junction. Laminin 332 is a skin basement membrane glycoprotein which was shown to be decreased in aged skin. We have previously demonstrated that the loss of laminin 332 leads to an upregulation of cholesterol biosynthesis genes and alters the skin’s lipid profile. We identified that this change is due to an impaired intracellular cholesterol transport. To understand the mechanism around cholesterol transport we have identified a panel of proteins of interest for further investigation. We investigated the role that laminin 332, actin and microtubule disruption have on both subcellular location of our proteins of interest and cholesterol transport. We have demonstrated that NPC2, Myo5b and e-cadherin are decreased in laminin α3 knockdown nTERT keratinocytes compared to shC controls. Interestingly, NPC1, a binding partner for NPC2, was shown to be increased in the laminin α3 knockdown nTERT keratinocytes. Normal levels and localisation of each protein can be restored by the addition of recombinant laminin 332. We have also demonstrated that in keratinocytes free cholesterol localises to Rab11a-positive vesicles which is a binding partner for Myo5b. By using actin- and microtubule-disrupting drugs, we have shown that it is the loss of actin that’s driving the observed phenotype. These findings suggest that the disrupted cholesterol transport and impaired barrier function in laminin α3 knockdown is due to lower levels of Myo5b and e-cadherin resulting from a disorganised actin cytoskeleton with a concomitant weakening of cell-cell contacts.
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